STABILIZATION OF RESVERATROL THROUGH …

STABILIZATION OF RESVERATROL THROUGH MICROENCAPSULATION AND INCORPORATION INTO FOOD PRODUCTS

BY

CLARISSA C. KOGA

DISSERTATION

Submitted in partial fulfillment of the requirements for the degree of Doctor of Philosophy in Food Science and Human Nutrition

with a concentration in Food Science in the Graduate College of the

University of Illinois at Urbana-Champaign, 2015

Urbana, Illinois

Doctoral Committee: Professor Keith Cadwallader, Chair Associate Professor Soo-Yeun Lee, Co-Director of Research Assistant Professor Youngsoo Lee, Co-Director of Research Assistant Professor Juan Andrade Professor Mario Ferruzzi, Purdue University

ii

ABSTRACT

There is an increasing prevalence of chronic disease and obesity in the United States.

Bioactive compounds which have been shown to prevent and alleviate disease states can aid in

decreasing this prevalence. The addition of these compounds to food products would help to

deliver these health benefits to consumers.

Challenges such as instability in environmental conditions and the digestive system, as

well as negative sensory properties of the bioactive compounds limits the addition of these

compounds to food products. Encapsulation is a processing technique that can help to

overcome these challenges and increase the range of food products into which the compounds

can be incorporated within. The long term goal of this research is to enhance stability and ease

of consumption of bioactive compounds by way of utilizing microencapsulation, which will

increase the consumption of these healthful ingredients.

This research utilized resveratrol, a polyphenol found in red grapes and wine, as a

model compound which encapsulation was applied in order to overcome the light instability and

bitterness of the compound. Resveratrol was encapsulated within a protein matrix through

spray drying. Encapsulated resveratrol within a sodium caseinate matrix had a higher UVA light

and digestive stability (0.63 trans:cis resveratrol ratio and 84%) than unencapsulated resveratrol

(0.49 trans:cis resveratrol ratio and 47%). In addition, the encapsulation of resveratrol

decreased the detection of the compound in comparison to unencapsulated resveratrol. The

resveratrol microcapsules were added to snack bars and gummies in order to show application

of the stabilized resveratrol. A group of consumers was identified who’s overall liking for the

food products were maintained with the addition of the encapsulated resveratrol.

The encapsulation-system approach developed in this research can be extended to

other bioactive compounds in order to increase stability and minimize negative sensory

properties of the target compounds, while delivering the health benefits of the compounds. This

iii

research serves as a basis upon which additional research can tailor the encapsulation-system

approach for the specific properties of the compound.

iv

ACKNOWLEDGMENTS

The journey of completing a doctoral degree has been filled with invaluable experiences

and lessons which cannot simply be taught in the classroom. For me, the best part of graduate

school was the people. I am so grateful to have been surrounded by many ambitious, talented,

and caring individuals.

Thank you to my advisors, Dr. Soo-Yeun Lee and Dr. Youngsoo Lee, for their guidance

and support in my research and personal development. You have pushed me to improve as a

person and as a scientist. I would also like to thank my doctoral committee: Dr. Juan Andrade,

Dr. Keith Cadwallader and Dr. Mario Ferruzzi. Thank you for your continued input and allowing

me to use your equipment. Also, thank you to all the past and present members of the Lee labs

for always being there for me through laughter and through the tough times. In addition, I would

like to thank my undergraduate advisor, Dr. Wayne Iwaoka, who encouraged me to pursue

higher education and helped me to see my potential. Lastly, thank you to my family back in

Hawaii for supporting me in every aspect of my life and believing in me. From the bottom of

heart, I would like to thank everyone who has been a part of this journey.

v

TABLE OF CONTENTS

CHAPTER 1: INTRODUCTION ...................................................................................... 1 1.1 Motivation ......................................................................................................................... 1 1.2 Project Objectives ............................................................................................................. 1

Objective 1 .......................................................................................................................... 1 Objective 2 .......................................................................................................................... 2

1.3 Outline of Thesis ............................................................................................................... 2 1.4 References ....................................................................................................................... 4

CHAPTER 2: LITERATURE REVIEW ............................................................................ 5 2.1 Introduction ....................................................................................................................... 5 2.2 Health Benefits ................................................................................................................. 5

2.2.1 Cardiovascular Disease ............................................................................................. 6 2.2.2 Cancer ....................................................................................................................... 6 2.2.3 Diabetes Mellitus ........................................................................................................ 7 2.2.4 Neurological Function ................................................................................................ 7 2.2.5 Lifespan ..................................................................................................................... 7 2.2.6 Obesity....................................................................................................................... 8

2.3 Anti-microbial and Anti-oxidant Functions ......................................................................... 8 2.4 Resveratrol Sources ......................................................................................................... 8

2.4.1 Resveratrol as a Defense Mechanism in Plants ......................................................... 9 2.4.2 Transgenic Fruit with High Resveratrol Content ......................................................... 9

2.5 Regulations .....................................................................................................................10 2.6 Challenges ......................................................................................................................10 2.7 Bioavailability and Bioaccessibility ...................................................................................11 2.8 Increasing Stabilization and Bioaccessibility ....................................................................12

2.8.1 Cyclodextrin Complexes ...........................................................................................12 2.8.2 Porous Microspheres ................................................................................................13 2.8.3 Cross-linked Chitosan ...............................................................................................14 2.8.4 Pectinate Beads ........................................................................................................14 2.8.5 Lipid Nanoparticles and Carriers ...............................................................................15 2.8.6 Double Emulsion .......................................................................................................15 2.8.7 Nanoencapsulation ...................................................................................................16 2.8.8 Niosomes ..................................................................................................................16 2.8.9 Encapsulation Combination .......................................................................................17

2.9 Increasing Solubility .........................................................................................................17 2.10 Protein Binding ..............................................................................................................17 2.11 Resveratrol Recovery ....................................................................................................18 2.12 Food and Non-Food Applications ...................................................................................19 2.13 Sensory Analysis ...........................................................................................................20 2.14 Conclusion .....................................................................................................................21 2.15 Figures and Tables ........................................................................................................22 2.16 References ....................................................................................................................27

CHAPTER 3: STABILITY AND BINDING OF TRANS-RESVERATROL ENCAPSULATED IN A PROTEIN MATRIX PRODUCED USING SPRAY DRYING ... 34

3.1 Abstract ...........................................................................................................................34 3.2 Introduction ......................................................................................................................35 3.3 Materials and Methods ....................................................................................................38

vi

3.3.1 Materials ...................................................................................................................38 3.3.2 Methods ....................................................................................................................39

3.3.2.1 Microcapsule Production....................................................................................39 3.3.2.2 High-performance Liquid Chromatography ........................................................39 3.3.2.3 Moisture Content and Water Activity Measurements ..........................................40 3.3.2.4 Morphology and Particle Size ............................................................................40 3.3.2.5 Microencapsulation Efficiency Measurements ...................................................40 3.3.2.6 Resveratrol Recovery Measurements ................................................................41 3.3.2.7 Fluorescence .....................................................................................................41 3.3.2.8 Fluorescence of Resveratrol and Ethanol ..........................................................41 3.3.2.9 Fluorescence of Solution with Spiked Resveratrol with Various Resveratrol

Concentrations ...................................................................................................42 3.3.2.10 Fluorescence of Microcapsules with Varying Resveratrol Concentrations ........42 3.3.2.11 Stern-Volmer Equation.....................................................................................43 3.3.2.12 Equation Based on Number of Resveratrol Binding Sites ................................43 3.3.2.13 UVA Stability Measurements ...........................................................................45 3.3.2.14 In-Vitro Digestion Test .....................................................................................46 3.3.2.15 Statistical Analysis ...........................................................................................47

3.4. Results and Discussion ..................................................................................................48 3.4.1 Moisture Content and Water Activity Measurements .................................................48 3.4.2 Morphology and Particle Size ....................................................................................48 3.4.3 Microencapsulation Efficiency ...................................................................................49 3.4.4 Resveratrol Recovery ................................................................................................49

3.4.4.1 Fluorescence .....................................................................................................50 3.4.4.2 Estimated Resveratrol Binding Using Stern-Volmer Equation ............................50 3.4.4.3 Effect of pH on Stern-Volmer Calculations .........................................................51 3.4.4.4 Estimated Resveratrol Binding Using Resveratrol Binding Sites Equation .........52 3.4.4.5 Compare the Resveratrol Binding Using Stern-Volmer Equation and

Resveratrol Binding Sites Equation ....................................................................52 3.4.4.6 Total Resveratrol Accounted For .......................................................................53 3.4.4.7 Binding of Resveratrol and Protein ....................................................................53

3.4.5 UVA Stability .............................................................................................................55 3.4.6 In-Vitro Digestions .....................................................................................................56

3.5 Conclusions .....................................................................................................................57 3.6 Figures and Tables ..........................................................................................................58 3.7 References ......................................................................................................................73

CHAPTER 4: EFFECT OF PLASTICIZERS ON STABILITY OF ENCAPSULATED RESVERATROL ........................................................................................................... 77

4.1 Abstract ...........................................................................................................................77 4.2 Introduction ......................................................................................................................78 4.3 Material and Methods ......................................................................................................80

4.3.1 Materials ...................................................................................................................80 4.3.2 Microcapsule Production ...........................................................................................80 4.3.3 Morphology and Particle Size ....................................................................................81 4.3.4 Moisture Content and Water Activity .........................................................................81 4.3.5 HPLC ........................................................................................................................82 4.3.6 UV Stability ...............................................................................................................82 4.3.7 Statistics ...................................................................................................................83

4.4 Results and Discussion ...................................................................................................83 4.4.1 Morphology and Particle Size ....................................................................................83

vii

4.4.2 Moisture Content and Water Activity .........................................................................84 4.4.3 UV Stability ...............................................................................................................84

4.5 Conclusions .....................................................................................................................86 4.6 Figures and Tables ..........................................................................................................87 4.7 References ......................................................................................................................92

CHAPTER 5: TASTE DETECTION THRESHOLDS OF RESVERATROL ................... 94 5.1 Abstract ...........................................................................................................................94 5.2 Introduction ......................................................................................................................95 5.3 Materials and Methods ....................................................................................................96

5.3.1 Encapsulated Resveratrol Production .......................................................................96 5.3.2 Sample Preparation ..................................................................................................97 5.3.3 Rating Method and R-index Measurement ................................................................99 5.3.4 Taste Detection Threshold Testing ............................................................................99 5.3.5 Testing of Additional Concentration Level ............................................................... 101 5.3.6 Post-Questionnaire ................................................................................................. 102 5.3.7 Data Statistical Analysis .......................................................................................... 102 5.3.8 Taste Detection Threshold Calculations .................................................................. 102

5.4 Results and Discussion ................................................................................................. 103 5.4.1 Taste Detection Threshold ...................................................................................... 103 5.4.2 Post Questionnaire ................................................................................................. 105

5.5 Conclusions ................................................................................................................... 106 5.6 Figures and Tables ........................................................................................................ 108 5.7 References .................................................................................................................... 111

CHAPTER 6: CONSUMER ACCEPTANCE OF BARS AND GUMMIES WITH RESVERATROL AND ENCAPSULATED RESVERATROL ...................................... 113

6.1 Abstract ......................................................................................................................... 113 6.2 Introduction .................................................................................................................... 114 6.3 Materials and Methods .................................................................................................. 116

6.3.1 Materials ................................................................................................................. 116 6.3.2 Encapsulated Resveratrol Production ..................................................................... 116 6.3.3 Preparing Samples ................................................................................................. 116 6.3.4 Bars ........................................................................................................................ 117 6.3.5 Gummies ................................................................................................................ 117 6.3.6 Resveratrol Dosage ................................................................................................ 117 6.3.7 Panelists ................................................................................................................. 118 6.3.8 Consumer Testing ................................................................................................... 118 6.3.9 Post-Questionnaire ................................................................................................. 119 6.3.10 Data Analyses ....................................................................................................... 119

6.4 Results and Discussion ................................................................................................. 120 6.4.1 Overall Liking .......................................................................................................... 120 6.4.2 Check-all-that-apply ................................................................................................ 121 6.4.3 Post-Questionnaire ................................................................................................. 123

6.5 Conclusion ..................................................................................................................... 124 6.6 Figures and Tables ........................................................................................................ 125 6.7 References .................................................................................................................... 141

CHAPTER 7: SUMMARY ........................................................................................... 143

viii

CHAPTER 8: APPENDICES ...................................................................................... 145 Appendix A: Apparent solubility of 180 mg Resveratrol/L in 1.2% Ethanol Solution ............. 145 Appendix B: Post-Questionnaire for Threshold Testing of Resveratrol ................................. 146 Appendix C: Ballot for Consumer Testing on Food Products with Resveratrol ..................... 148 Appendix D: Post-Questionnaire for Consumer Testing on Food Products with Resveratrol 150

1

CHAPTER 1: INTRODUCTION

1.1 Motivation

In the United States, more than 1/3 of adults are obese which poses concern as many

chronic diseases, such as heart disease, stroke, diabetes, cancer, liver problems, and

respiratory problems, are related with obesity [1, 2]. The overall medical cost in the United

States related to obesity in 2008 was $147 billion dollars [2].

Healthy eating habits and regular exercise can help to combat obesity and rising

prevalence of chronic disease in the United States. With this being said, it is often difficult for

people to change their lifestyles. Therefore, the addition of healthful compounds to food

products can provide the health benefits in easy-to-consume foods and provide a convenient

means for the compounds to be delivered to consumers.

Resveratrol was a compound of interest due to its association with beneficial effects on

heart disease, cancer, diabetes, and neurological function [3-6]. Some of the challenges with

the incorporation of resveratrol into food products are instability in light, bitterness, and low

bioaccessibility through the human digestive system [7-9]. The long term goal of this research

is to enhance stability and ease of consumption of bioactive compounds by way of utilizing

microencapsulation, which will increase the consumption of these healthful ingredients. The

central hypothesis is microencapsulation of resveratrol within a protein matrix will affect

stability and sensory properties of the compound.

1.2 Project Objectives

Objective 1

The first objective of this research was to stabilize trans-resveratrol in the presence of

light and through the human digestive system. A common processing technique and cost-

effective encapsulation material would be desirable in order to make scale-up and application in

the food industry feasible. The working hypothesis for this objective was the use of

2

microencapsulation will increase the stability of resveratrol as evaluated through UV light and in-

vitro digestion testing.

Objective 2

The second objective of this research was to evaluate the sensory properties of the

stabilized resveratrol. Taste is one of the most important considerations when consumers are

purchasing a product [10]. Even if a product contains a healthful compound, it needs to have a

high acceptability in order for consumers to repeatedly purchase the product. The working

hypothesis for Objective 2 was the incorporation of resveratrol into a microcapsule will increase

the taste detection threshold of the compound, and further, not compromise consumer

acceptance of food products with encapsulated resveratrol.

1.3 Outline of Thesis

Chapter 2 is a literature review that provides an overview of resveratrol, associated

health benefits, regulations, challenges with incorporation into food products, binding between

resveratrol and protein, resveratrol recovery, bioaccessibility, processing methods to stabilize

resveratrol, and sensory properties of the compound.

Chapter 3 evaluates the stability of resveratrol in the four resveratrol microcapsule

formulations under UVA light and in-vitro digestion testing. Two proteins (whey protein

concentrate and sodium caseinate) and the effect of fat within the encapsulation matrix were

compared as wall materials in the microcapsules. In addition, the quantification of binding

between resveratrol and protein was explored. Resveratrol recovery from the microcapsules

was limited, thereby, prompting interest in the investigation of the interaction between

resveratrol and protein which may limit the ability for resveratrol to be extracted from the

microcapsules. It was thought that binding between resveratrol and protein plays a significant

role in stability of the compound.

3

Chapter 4 investigates the effect of plasticizers in the resveratrol microcapsule

formulation in terms of morphology of the microcapsules and UV stability of resveratrol. The

effect of plasticizer concentration and the use of protein denaturation on resveratrol stability

within the microcapsule was also studied in terms of UV stability.

Chapter 5 compares the taste detection threshold of resveratrol and resveratrol

encapsulated within a protein matrix. The R-index measure by the signal detection rating

method in comparison to the noise and signal sample was utilized in this study. Resveratrol

was tested in 6-concentration levels that differed in 3-fold increments, ranging from 2.2-540

mg/L for unencapsulated resveratrol and between 4.4-1080 mg/L for encapsulated resveratrol.

Chapter 6 assesses consumer acceptance of bars and gummies with added resveratrol

microcapsules and unencapsulated resveratrol. Resveratrol was added to the food products at

concentrations of 10 and 40 mg/serving. These levels of resveratrol have been shown to

provide health benefits in humans [4, 11]. Panelists rated the overall liking of each product

along with indicating which attributes they liked and disliked about the products. This testing

gives indication of consumer perception of products with added resveratrol.

Chapter 7 concludes this dissertation with a summary of research findings, implications

to the food industry, and explanation of future research.

4

1.4 References

1. Ogden, C.L., et al., Prevalence of childhood and adult obesity in the united states, 2011-2012. JAMA, 2014. 311(8): p. 806-814.

2. Centers for Disease Control and Prevention, Obesity: Halting the epidemic by making health easier at a glance. 2011.

3. Wang, Z., et al., Effects of red wine and wine polyphenol resveratrol on platelet aggregation in vivo and in vitro. International Journal of Molecular Medicine, 2002. 9: p. 77-80.

4. Brasnyó, P., et al., Resveratrol improves insulin sensitivity, reduces oxidative stress and activates the akt pathway in type 2 diabetic patients. Br J Nutr, 2011. 106(3): p. 383-389.

5. Miura, D., Y. Miura, and K. Yagasaki, Hypolipidemic action of dietary resveratrol, a phytoalexin in grapes and red wine, in hepatoma-bearing rats. Life sciences, 2003. 73(11): p. 1393-1400.

6. Kim, D., et al., Sirt1 deacetylase protects against neurodegeneration in models for alzheimer's disease and amyotrophic lateral sclerosis. The EMBO journal, 2007. 26(13): p. 3169-3179.

7. Walle, T., et al., High absorption but very low bioavailability of oral resveratrol in humans. Drug metabolism and disposition, 2004. 32(12): p. 1377-1382.

8. Gaudette, N. and G. Pickering, Sensory and chemical characteristics of trans‐resveratrol‐fortified wine. Australian Journal of Grape and Wine Research, 2011. 17(2): p. 249-257.

9. Vian, M.A., et al., Simple and rapid method for cis-and trans-resveratrol and piceid isomers determination in wine by high-performance liquid chromatography using chromolith columns. Journal of Chromatography A, 2005. 1085(2): p. 224-229.

10. Stanton, J., Taste: After 40 years, still job one. Food Processing, 2013. 74(9): p. 98. 11. Ghanim, H., et al., An antiinflammatory and reactive oxygen species suppressive effects

of an extract of polygonum cuspidatum containing resveratrol. Journal of Clinical Endocrinology & Metabolism, 2010. 95(9): p. E1-E8.

5

CHAPTER 2: LITERATURE REVIEW

2.1 Introduction

Resveratrol (3,4’,5-trihydroxystilbene) found in red grapes and peanuts has been shown

to have numerous health benefits associated with heart disease, cancer, diabetes, neurological

function and other biological functions [1-6]. It has also been shown to have anti-microbial and

anti-oxidant properties [7, 8]. The trans-isomer of resveratrol is the form which has been

associated with health benefits, but instability in light, insolubility in aqueous solutions, low

bioaccessibility and bitterness limit the range of products into which the compound can be

incorporated [9-12].

Various processing methods are being used to overcome the challenges of resveratrol

incorporation into foods. Processing methods can aid in the innovation of enhancing stability

and decreasing negative sensory properties of resveratrol. Innovative processing methods can

aid in the incorporation of resveratrol into foods with the long term goal of providing the health

benefits of the compound to consumers. In terms of stability, prior research was mainly focused

on the encapsulation of resveratrol in order to limit isomerization of the compound. The

encapsulation techniques which have been explored in prior research are cyclodextrin

complexes, porous microspheres, cross-linked chitosan, pectinate beads, lipid nanoparticles

and carriers, double emulsions, nanoencapsulation, and niosomes [13-19]. In addition,

processing methods to increase the solubility of resveratrol through addition of stevioside and

application to food systems such as hazelnut paste and edible films have been researched [20-

22]. There are limited published studies available on the sensory properties of resveratrol.

2.2 Health Benefits

Extensive research supports resveratrol is associated with health benefits related to

heart disease, cancer, diabetes, neurological function, and other biological activities [1-6].

6

Table 2.1 summarizes the extensive body of literature on various health benefits according to

the type of animal model used in the research.

2.2.1 Cardiovascular Disease

Research findings support that resveratrol lowered platelet aggregation in platelet-rich

plasma and isolated human platelets [23, 24]. It was also able to lower total cholesterol and

low-density lipoprotein (LDL) levels in hypercholesterolaemic rats [1]. In addition, resveratrol

was able to improve vascular function by decreasing total plasma cholesterol and cholesterol in

lipoprotein fractions in mice [25]. An increased recovery at reperfusion and significant

vasodilation which indicates cardioprotection was also found in rats [26]. The effects of

resveratrol on heart disease were also investigated in guinea pigs and found to increase cardiac

DT-diaphorase and catalase activity. It also resulted in relaxation of arteries which supported

the cardioprotective effects of the compound [27, 28]. In rabbits, resveratrol intake helped to

suppress atherosclerosis and ADP-induced platelet aggregation, indicating prevention of

coronary heart disease [24, 29]. Furthermore, a dose dependent inhibition of platelet

aggregation and increased cerebral blood flow were reported when resveratrol was

administered to humans, which indicated a decreased risk of cardiovascular disease [3, 24, 30].

2.2.2 Cancer

A second health benefit of resveratrol is its ability to prevent cancer and slow tumor

growth. It is thought that resveratrol can inhibit carcinogenesis at multiple stages. Resveratrol

has been tested against ovarian carcinoma cell lines, and it was found to inhibit the proliferation

and survival of these cells; thereby causing autophagocytosis that helps to maintain

homeostasis in cells [31]. In other cell lines, resveratrol helped to decrease basal production of

PGE2, inhibit PMA-mediated activation of protein kinase C and COX-2, and decrease ornithine

decarboxylase activity [32-34]. These changes in biomarkers were indications of inhibiting

cancer proliferation. The administration of resveratrol to rat and mice models helped to prevent

initiation and progression of tumor growth, even decreasing tumor size and increasing survival

7

of the animal [1, 35-46]. Healthy adults were given 0.5, 1, 2.5 or 5 g of resveratrol/day and a

decrease in IGF-1 and IGFBP-3 was observed, which indicated chemoprotection [47].

2.2.3 Diabetes Mellitus

Thirdly, resveratrol has been associated with the prevention and treatment of diabetes

mellitus. Resveratrol has been found to have an anti-hyperglycemic effect and also reduced

glycosylated hemoglobin that further confirmed a reduction in blood sugar [48-51]. Resveratrol

facilitated intracellular glucose transport even in the absence of insulin [52]. Furthermore,

insulin sensitivity was increased in healthy rats and also in insulin-resistant mice on a high-fat

diet after the consumption of resveratrol [51, 53]. In diabetic males, resveratrol has been shown

to decrease blood glucose and insulin resistance, showing positive effects on diabetes [4, 5].

2.2.4 Neurological Function

The fourth health benefit of resveratrol is an increase in neurological function. It has

been found that resveratrol helped to reduce neuronal cell death and neurotoxicity in an

Alzheimer’s mouse model [2]. Another study found resveratrol was able to reduce amyloid

plaques in mice which is thought to be associated with decreased glutathione and increased

cysteine [54]. Furthermore, other biomarkers such as NF-kappaB p65 expression, and

decreased malondialdehyde levels are thought to be related to improved neurological function

[55-57]. Research have shown that resveratrol intake stimulated Sir1, that inhibited nuclear

factor κB signaling which helped to prevent β-amyloid toxicity and showed a therapeutic effect

against Alzheimer disease [58].

2.2.5 Lifespan

Not only has resveratrol been associated with the prevention of disease states but also

an increase in lifespan. Sirtuins are a group of genes associated with the aging pathway;

therefore, the up-regulation of these genes is correlated with increased lifespan. In yeast,

resveratrol has been shown to activate Sir2 and increase lifespan by 70% [59]. Another study

has paralleled studies in yeast finding that resveratrol was able to increase lifespan in flies, fish,

8

worms, and mice [60-63]. Lagouge and others (2006) found that resveratrol intake prevented

diet-induced obesity, increased aerobic capacity and inhibited muscle fatigue in mice [64].

2.2.6 Obesity

Resveratrol has also been shown to have positive effects on obesity. A resveratrol

supplementation of 0.4% to mice on a high-fat diet was shown to decrease body weight gain,

visceral fat, and plasma biomarkers related to adipogenesis and inflammation [65]. In non-

human primates, it was found that 200 mg resveratrol/kg body weight/day reduced energy

intake by 13% and increased resting metabolic rate by 29% [66]. It is hypothesized that

resveratrol can increase satiety thereby decreasing energy intake and increasing energy

expenditure.

2.3 Anti-microbial and Anti-oxidant Functions

Additional functional properties of resveratrol include its ability to serve as anti-microbial

and anti-oxidant agents. At concentrations of 11 and 22 mg/L, resveratrol was shown to inhibit

the growth of Saccharomyces cerevisiae, Penicillium expansum, and Aspergillus niger [7]. The

effectiveness of resveratrol against HIV has also been shown [67, 68]. The ability of resveratrol

to increase oxidative stress tolerance was studied in yeast. The yeasts were exposed to

oxidative agents such as CCl4 and H2O2. In the presence of resveratrol, there was a significant

increase in survival rate and reduction in lipid peroxidation [8].

2.4 Resveratrol Sources

Resveratrol is naturally found in low concentrations in some fruits, wine, and nuts. The

concentrations of resveratrol in red grapes, red wine, white wine, peanuts, and blueberries are

0.050 mg/100 g, 0.002-0.653 mg/L, trace-0.100 mg/L, 0.002-.0179 mg/100 g, and trace-0.003

mg/100 g, respectively [69-72]. The higher resveratrol content in peanuts than red grapes may

be due to the high water content in grapes in comparison to peanuts. Therefore, it appears

there is a lower concentration of resveratrol in red grapes.

9

2.4.1 Resveratrol as a Defense Mechanism in Plants

In plants, resveratrol may be produced as a defense mechanism to fungal invasion and

other physical damage [71]. This defense mechanism has been specifically observed in

peanuts that may provide an explanation for the wide range of resveratrol concentration found in

this food source. When peanut seedling leaves were exposed to UVC irradiation, it was found

that endogenous resveratrol increased significantly within 24 hours of exposure [73]. The

effects of UVC irradiation was also explored in grape leaves [74]. Endogenous resveratrol was

shown to increase 180-196 fold in comparison to the control (not exposed to UVC light) at 16 hr

and 24 hr. Stilbene synthase, which is an important enzyme in the resveratrol biosynthesis, was

also increased in the grape leaves after UVC exposure. Aluminum chloride has also been used

to enhance accumulation of resveratrol in grape vine leaves [75]. All concentrations of

aluminum chloride which were tested, 7-90 mM of aluminum chloride, were shown to induce

resveratrol production. In addition, resveratrol has been shown to be produced as a defense

response to Botrytis in grapevines. The level of resveratrol production was correlated to the

level of pathogen infection [76]. Resveratrol has also been shown to be produced when

grapvines were infected with mildew disease [77].

2.4.2 Transgenic Fruit with High Resveratrol Content

The concentration of resveratrol shown to have biological effects in humans is higher

than that naturally found in plants. Therefore, transgenic produce have been developed in order

to increase resveratrol concentration in natural sources of resveratrol. A stilbene synthase

gene, associated with resveratrol production in fruits and vegetables, has been overexpressed

in order to increase resveratrol content in lettuce [78]. Resveratrol content in the lettuce

reached 56.40 ± 5.52 ug/g. Stilbene synthase gene has also been used to increase resveratrol

glucoside, a resveratrol derivative in apples [79].

10

2.5 Regulations

Currently, resveratrol is not regulated as a drug in the United States and instead is

considered a dietary supplement [80]. The Dietary Supplement Health and Education Act of

1994 made it the responsibility of manufacturers to determine the safety of compounds such as

resveratrol. Under this law, the manufacturers can provide basic information about the

mechanism by which the supplement affects “the structure or function of humans” but claims

about the treatment or prevention of diseases cannot be made [81, 82]. Little is known about

the amount of resveratrol intake that is considered safe.

The safety of resveratrol consumption is a viable concern when considering the

incorporation of the compound into food products. No adverse effects were seen in rats when

fed 50-700 mg resveratrol/kg bw/day for 28-90 days [83]. Other research found a decrease in

white blood cells when rats were fed 450 mg resveratrol/kg bw/day for 56 days [53]. No serious

effects were seen when healthy adults were given a 0.5-5 g single dose, but minor effects such

as increased bilirubin and alanine aminotransferase were seen at a 1 g dosage [84]. Headache

and dizziness were also seen in healthy adults when given a total of 150-900 mg resveratrol/day

in small dosages every 4 hours [85]. The effects of resveratrol in rats and humans have been

shown to not be serious, and the dosages that have been used in safety testing are well above

those which have been shown to have biological effects.

2.6 Challenges

The incorporation of resveratrol into food products would help to deliver the health

benefits of the compound to consumers, but resveratrol is unstable in certain environmental

conditions such as light, pH and heat. The activation energy to isomerize trans-resveratrol to

cis-resveratrol has been found to be ~3.7 kcal/M [72]. This activation energy is relatively low as

the activation energy to go from H2O2 H2O + O2 was ~10 kcal/M [86]. The biggest challenge

behind the addition of resveratrol to food products is instability of resveratrol in the presence of

11

light [9, 87]. After one hour of exposure to light, 80-90% of trans-resveratrol (bioactive) was

converted to cis-resveratrol (bio-inactive) [87]. Another study found that about 90% of trans-

resveratrol was converted to cis-resveratrol after UV exposure for 120 min at 366 nm [9].

Trans-resveratrol was most stable between pH 5-8 as the isomerization was slowest in this pH

range [88]. Heat is another factor that can lead to instability of trans-resveratrol. Exposure to

190°C for 18 min has been shown to degrade 17-46% of resveratrol in blueberries and

biliberries [70].

Another challenge with resveratrol incorporation into food products is its limited solubility

in both aqueous and lipid solutions. The reported amount of soluble resveratrol was 0.03

mg/mL in water [10] and 0.18 mg/mL in coconut oil [89]. It is soluble in ethanol at 50 mg/mL and

also in dimethyl sulfoxide at 16 mg/mL [10]. Additionally, relative humidity has not been found

to affect isomerization of trans-resveratrol significantly at 25°C when relative humidity was

increased from 75% to 90% [90].

2.7 Bioavailability and Bioaccessibility

An important consideration when processing resveratrol for incorporation into food

products is the bioavailability and digestibility of the resveratrol. When resveratrol was

administered orally through a drink, serum resveratrol levels peaked at about 30 minutes post-

consumption and less than 2% of resveratrol was found in the trans-isomer [91]. Other

research showed that resveratrol glucuronides and sulfates were mainly found in the plasma

and were detected for a prolonged amount of time. Walle and others (2004) found that in

addition to glucuronic acid and sulfate metabolites, the hydrogenation of aliphatic double bonds

also occured. The majority of the metabolites were found in the urine [92]. These findings

suggested that the resveratrol was partly metabolized in the small intestine and then distributed

to various parts of the body in conjugated form [11]. This theory was supported by another

research study as 50% of trans-resveratrol and derived metabolites administered to pigs was

12

found in the jejunum and ileum of the small intestine [93]. Resveratrol metabolites have been

found to accumulate in the liver probably due to partial metabolism of the compound in the liver

and also the transport of metabolites from the intestine. In the kidneys, resveratrol levels

gradually decreased with time that suggested that renal excretion is one of the predominant

means of elimination [11]. Therefore, these findings support that resveratrol readily breaks

down in the body resulting in low bioaccessibility and bioavailability.

2.8 Increasing Stabilization and Bioaccessibility

Research has explored various ways to overcome the challenges of resveratrol related

to instability and low bioaccessibility. The main method of stabilizing resveratrol is

encapsulation that helps to prevent isomerization and maintain the bioactivity.

2.8.1 Cyclodextrin Complexes

Cyclodextrin complexes are the most common form of resveratrol stabilization. The

number of glucose residues connected by α(14) glycosidic bonds can range from six to eight.

Cyclodextrins can form inclusion complexes with a wide range of compounds as they have a

hydrophobic inner cavity and hydrophilic capsule walls. In this way, resveratrol can form an

inclusion complex with the hydrophobic cavity thereby protecting resveratrol [13]. In all the

studies involving encapsulation of resveratrol by cyclodextrin complexes, antioxidant testing was

the primary means to evaluate the microcapsules. β- and maltosyl-β-cyclodextrins have been

used to form complexes with resveratrol and testing supported that biological activities were

maintained. Oxidation of resveratrol by lipoxygenase was tested for both cyclodextrins [13].

Mantegna and others (2012) formed β-cyclodextrin and resveratrol complexes through

ultrasound-assisted extraction from Polygonum cuspidatum. The complexation helped to

increase antioxidant capacity that was tested through DPPH radical scavenging and ORACFL

test [94]. The cyclodextrins also helped to increase dispersibility and reduced time needed to

extract resveratrol from the root. Unmodified β-cyclodextrin was thought to be unsafe due to

13

nephrotoxicity. Therefore, the use of modified hydroxypropyl-β-cyclodextrin as a complexation

agent of resveratrol has also been explored [95]. This modified cyclodextrin has been found to

form a similar 1:1 inclusion complex, but with a larger inclusion constant that indicates a greater

inclusive potential. The solubility and antioxidant efficacy of β-cyclodextrins and hydroxypropyl-

β-cyclodextrins were investigated. The resveratrol-cyclodextrin complexes had a higher

solubility and similar antioxidant activity to free resveratrol [95]. The use of hydropropyl-β-

cyclodextrin for resveratrol complexation decreased oxidation of the compound by free radicals

and increased antioxidant activity [96]. The cytotoxicity of resveratrol complexed with β-

cyclodextrin or 2-hydroxypropyl-β-cyclodextrin was compared [97]. The complex of resveratrol

and 2-hydroxypropyl-β-cyclodextrin showed a higher cytotoxicity against two cancer cells in

comparison to β-cyclodextrin complexation with resveratrol. Resveratrol was incorporated into

the hydrophobic cavity of the cyclodextrin, therefore, hydrophobic interactions occurred between

the compound and cyclodextrin [13]. In comparison, other encapsulation methods of

resveratrol, such as pectinate beads, emulsions, and nanoparticles, utilize physical entrapment

of resveratrol within a wall structure.

2.8.2 Porous Microspheres

Prior research utilized porous polymeric microspheres for the stabilization of resveratrol,

looking specifically at the wetting time and cyano-functional groups [14]. The polymer particles

are an effective means to stabilize resveratrol because they scatter light that limits light

exposure to the compound. Additionally, resveratrol was maintained within the microsphere

through hydrogen bonding of the compound and cyano-functional groups of the particles. The

particles were produced by first forming monodisperse porous polymer particles containing

cyano groups by dispersion polymerization. Then, the porous particles underwent a wetting

period of 24 hours before resveratrol was immobilized within the porous particles by dropping an

ethanol and resveratrol mixture into the porous particle solution. The results showed that the

encapsulation helped to preserve 93% of the antioxidant activity and maintained bioactivity for 5

14

weeks. Confocal laser scanning microscopy analysis confirmed that the resveratrol was evenly

distributed within the porous particle, and it was hypothesized that hydrogen bonding helps

provide stability to the compound.

2.8.3 Cross-linked Chitosan

Cross-linked chitosan microspheres are another method that has been utilized to

increase resveratrol stability. Chitosan is commonly used in the food industry and is a desirable

material for stabilization because it is non-toxic, bio-compatible and able to form films. Cross-

linkers were used for chitosan microspheres to increase stability and control the release of the

bioactive compounds [15]. Vanillin is an ideal cross-linker because it is generally regarded as

safe and has bioactive properties [98, 99]. The microspheres were formed by an emulsion and

chemical cross-linking method. Encapsulation efficiency measurements showed 93.68%

efficiency for the chitosan microspheres with vanillin cross-linking. The microspheres were

exposed to UV light for 1 hr and temperatures of 60°C and 70°C for 15 days. The results

support that the chitosan microspheres were able to preserve the resveratrol retention [15].

2.8.4 Pectinate Beads

Calcium-pectinate beads have been used to stabilize resveratrol in order to delay the

release of the compound from the capsule [16, 100]. This method was selected in order to

achieve colon-specific delivery of resveratrol for the treatment of lower gastro-intestinal

disorders. The capsules were formed through ionotropic gelation where a resveratrol and pectin

solution was crosslinking into a calcium, chloride, and polyethyleneimine solution. The beads

were then washed with water and dried. Various parameters were altered to form capsules with

a delayed drug release profile and high encapsulation efficiency. It was found that 0.1%

polyethyleneimine solution with 2 hour crosslinking time and 0.2% polyethyleneimine with 0.5

hour crosslinking time showed an optimum drug release profile and high encapsulation

efficiency.

15

Zinc-pectinate beads have also been used to encapsulate resveratrol [101]. It was

found that zinc-pectinate beads had a better delayed release than calcium-pectinate beads.

The release of resveratrol from the beads followed zero-order kinetics, and the beads were

stable at room temperature and 4°C.

2.8.5 Lipid Nanoparticles and Carriers

Solid lipid nanoparticles and nanostructured lipid carriers have also been utilized to

encapsulate and protect resveratrol [102]. High shear homogenization was used to produce the

nanoparticles. Both particles were able to decrease reactive oxygen species, but nanostructure

lipid carriers were able to permeate deeper into the skin. Solid lipid nanoparticles have also

been produced through solvent diffusion-solvent evaporation [103]. Encapsulation efficiency of

these particles was 88.9 ± 3.1%, and the in-vitro drug release was up to 120 hours.

2.8.6 Double Emulsion

A water-in-oil-in-water double emulsion is where an aqueous solution is first dispersed in

oil to form the primary emulsion which is then re-emulsified in water. These types of double

emulsions have been utilized to stabilize resveratrol. A benefit of multiple emulsions is that it

provides protection against oxidation of the target compound. The concentration of resveratrol

used was 200 mg/L in order to ensure dissolution of the compound. It was found that less than

10% of the initial resveratrol content migrated to the external aqueous phase after 2 weeks of

storage [17]. Response surface methodology was used to find the optimum ratio of primary and

secondary emulsifiers which were as follows: 1:2 ratio of core material to coating material,

1.25% w/v primary emulsifier concentration, 1:1.23 ratio of W/O emulsion to secondary coating

material, and 1.21% w/v secondary emulsifier concentration [104]. Whey protein was found to

be the most stable secondary coating material. Further research can investigate the use of

higher concentrations of resveratrol in emulsions and different emulsion materials in order to

minimize the passive diffusion of resveratrol into surrounding aqueous phase.

16

2.8.7 Nanoencapsulation

Nanoencapsulation is when a bioactive compound is entrapped within a nano-sized

capsule or emulsion. Resveratrol nanoemulsions have also been utilized to increase stability

and bioavailability of the compound [18]. Nanoencapsulation is beneficial because the small

size is thought to increase cellular uptake. The emulsions were composed of peanut oil and

0.01% by weight resveratrol processed with high-pressure homogenization. The chemical

stability of the emulsions was tested under different storage temperatures of 4ºC, 30ºC, and

55ºC for 30 days. There was no cis-resveratrol detected in the encapsulated samples. Storage

at 4ºC degraded about 50% of resveratrol but at 30ºC and 55ºC, resveratrol was stable. With

an exposure to UV-C light for 2 hours, the nanoemulsions showed a decreased amount of cis-

resveratrol formation and a lower rate of degradation. In addition, in-vitro testing of the stomach

and small intestine processes showed that resveratrol nanoemulsions stabilized resveratrol and

preserved the trans-isomer throughout these processes. Nanoparticle system of resveratrol

have also been utilized to increase the solubility while increasing the hepatoprotective effect

[105]. Positive effects such as reduction in oxidative stress and inflammatory cytokines were

observed from the nanoparticle system.

2.8.8 Niosomes

Niosomes are non-ionic surfactant vesicles and another means by which resveratrol has

been stabilized with a controlled release of the compound. Niosomes were chosen for research

because they can encapsulate hydrophilic compounds in the aqueous layer and lipophilic

compounds on the vesicular membrane. In addition, niosomes have a relatively low cost and

high chemical stability in comparison to liposomes [19]. Span 60 and Span 80-cholesterol were

the encapsulation material in the niosomes. Span 80 niosomes had low entrapment efficiency

while Span 60 niosomes had a higher entrapment efficiency and slower rate of release.

Stability testing of the niosomes by light backscattering analysis showed that the Span 80

niosomes are more stable than the Span 60 niosomes [19].

17

2.8.9 Encapsulation Combination

Resveratrol has been encapsulated in combination with other bioactive ingredients. An

oil-in-water emulsion was utilized to enable the co-delivery of compounds that differ in solubility:

resveratrol, tributyrin and fish oil [106]. The emulsion was stabilized by heated milk protein,

glucose and modified resistant starch. This study forms the basis for the incorporation of

various bioactive ingredients into a single product which can deliver similar or synergistic health

benefits. The stability of multiple compounds within an emulsion was not compared to a single

compound, but future research can investigate this comparison.

2.9 Increasing Solubility

Resveratrol has a limited solubility in aqueous solutions [10]. Stevioside has been

added to resveratrol in order to increase the solubility of resveratrol in aqueous solutions [20]. It

is hypothesized that there is an interaction between the hydrophobic core of the stevioside and

the resveratrol thereby enhancing the solubility of the resveratrol. The resveratrol and

stevioside complex was 45 times more soluble in aqueous solution compared to the pure

compound, and the addition of the complexes increased oxidative stability of soy protein

emulsions.

2.10 Protein Binding

Resveratrol has been shown to bind with protein, and this interaction can further

enhance stability of the compound. It has been found that resveratrol interacts with β-

lactoglobulin in a 1:1 ratio. This binding was not able to completely prevent the conversion of

trans to cis-resveratrol, but it was capable of delaying the conversion. In addition, the

interaction helped to increase the solubility of resveratrol [107]. The binding between

resveratrol and β-lactoglobulin has been found to be on the outer surface of the ligand near

Trp19-Arg124. The complex was stable at acidic pH with a binding constant of ~104 M-1 [108].

18

Binding between resveratrol and proteins (sodium caseinate and whey protein) have

been investigated [109, 110]. Fluorescence has been utilized to explore binding of resveratrol.

For sodium caseinate, the binding constant was found to be 3.7-5.1 x 105 M-1. Hydrogen

bonding and hydrophobic interaction are thought to be responsible for the static and dynamic

binding between resveratrol and sodium caseinate. The fraction of binding sites available for

complexation was 1.20 at 25°C. For whey protein, the binding constant was between 1.7 x 104

– 1.2 x 105 M-1. Whey protein and resveratrol formed a 1:1 complex, which did not affect the

secondary structure of resveratrol. Dipole-dipole and Van der Waal interactions were thought to

be responsible for the binding between resveratrol and whey protein.

2.11 Resveratrol Recovery

Various means to increase stability of resveratrol have been developed therefore it is

necessary to find methods to characterize the complexes or microcapsules that are formed.

Recovery and efficiency are currently used for characterization, but different methods and

calculations are used across research studies, making it difficult to cross compare the results.

Nanoparticle system using polyvinyl alcohol and a cationic copolymer has been used to

encapsulate resveratrol [105]. Resveratrol recovery was 96.3% and defined as the actual

amount of resveratrol in the nanoparticle system compared to the theoretical maximum of

resveratrol in the nanoparticle system. Ethanol was used to extract resveratrol extensively from

vanillin cross-linked chitosan microspheres, and the resveratrol recovery was 93.68% [15].

Resveratrol recovery was also measured in resveratrol liposomes in which free resveratrol was

first removed by dialysis and then 0.025% non-ionic surfactant, Triton X-100, was used to

extract the resveratrol [103]. The resulting recovery was about 76%. The decreased amount of

recovery may be because dialysis was first used prior to the recovery analysis. In calcium-

pectinate beads with resveratrol, the compound was extracted by pectinase enzyme and

methanol was added to increase the solubility of the resveratrol [16]. Resveratrol recovery in

19

the calcium-pectinate beads was between 60-98%, depending on the various factors tested

(cross-linking time, pectin:resveratrol, and polyethyleneimine concentration).

Encapsulation efficiency is often used to compare the effectiveness of encapsulation

methods. This method is an indirect measurement of resveratrol content in the microcapsule as

it takes into account the amount of resveratrol that is easily washed from the surface of the

microcapsules rather than the amount of resveratrol extracted from the microcapsule. When

resveratrol was incorporated in liposomes, the technique used to produce the liposomes had a

significant effect on encapsulation efficiency, which is explained by the following equation:

Encapsulation efficiency = (𝑟𝑒𝑠𝑣𝑒𝑟𝑎𝑡𝑟𝑜𝑙 𝑙𝑜𝑎𝑑−𝑟𝑒𝑠𝑣𝑒𝑟𝑎𝑡𝑟𝑜𝑙 𝑖𝑛 𝑠𝑢𝑝𝑒𝑟𝑛𝑎𝑡𝑎𝑛𝑡

𝑟𝑒𝑠𝑣𝑒𝑟𝑎𝑡𝑟𝑜𝑙 𝑙𝑜𝑎𝑑) (eq 1.1)

Liposome prepared through sonication had an encapsulation efficiency of 44-56% and

those prepared by extrusion had an encapsulation efficiency of 92-96% [111]. Also, the

encapsulation efficiency of resveratrol nanoparticles in a methoxy poly(ethylene glycol)-

poly(caprolactone) matrix was found to be about 91% [112]. When resveratrol was

encapsulated in zinc-pectinate or calcium-pectinate, the encapsulation efficiency was relatively

high, between 97-99% [100, 101]. In comparison, resveratrol in solid lipid nanoparticles had an

encapsulation efficiency between 64-89%, and nanostructured lipid carriers had an

encapsulation efficiency between 65-77% [113]. Differences in encapsulation efficiency may be

due to the use of different processing techniques, wall materials, and method of resveratrol

extraction.

2.12 Food and Non-Food Applications

The ultimate goal is to incorporate resveratrol into food products, but there is very limited

research which shows application of resveratrol. One example of this incorporation for

functionality within a food matrix is the nanoencapsulated resveratrol was incorporated into

hazelnut paste in order to prevent oxidation of the paste [114]. The results showed that the

oxidation of hazelnut paste was reduced through the addition of encapsulated resveratrol.

20

Another study investigated resveratrol incorporation into edible film formation. Chitosan and

methylcellulose films were both used to incorporate the resveratrol. The addition of resveratrol

altered the film properties such as decreased ability to stretch and glossiness, and increased

resistance to fracture, opaqueness, and antioxidant activity [22].

Resveratrol has also been utilized for non-food application. Poly(L-lactic acid) films have

been formulated with various levels of both α-Tocopherol and resveratrol. The goal was to

develop a functional membrane which could be used for controlled release of antioxidants in

packaging. The equilibrium time for the diffusion of the antioxidants into ethanol decreased with

increased temperature. The addition of the antioxidants to the film resulted in a decreased

glass transition and melting temperature and increased thermal stability [115, 116]. These

findings are significant because they provide a model for incorporation of antioxidants into

packaging in order to reduce negative effects such as oxidation of the contents within the

packaging through chelation of the free radicals.

2.13 Sensory Analysis

There is limited research in the sensory attributes of resveratrol. One study investigated

the sensory and chemical characteristics of Riesling and Cabernet Sauvignon wines fortified

with two concentrations of trans-resveratrol: 20 and 200 mg/L. Analysis was completed at

various time points up to 58 weeks after bottling. The Riesling wine fortified with 20 mg/L trans-

resveratrol had a significantly higher bitterness than the unfortified control, but the highest

bitterness rating was the Riesling wine fortified with 200 mg/mL trans-resveratrol [12]. Another

research study identified the trans-resveratrol concentrations and sensory scores of various

wines. The results show there was no direct correlation between the trans-resveratrol

concentrations and the sensory scores of the wines, which were general ratings [117]. If

specific attributes were evaluated individually, then significant differences might have been

correlated with varying trans-resveratrol content.

21

2.14 Conclusion

The health benefits of resveratrol provide a valid rationale to add the bioactive form of

resveratrol into food products in order to disseminate the health benefits to the consumers.

Current research is investigating innovative approaches to overcome the challenges of

resveratrol incorporation into food products. Most of these approaches are not suitable for

mass production and have not been applied to commercial products. Future research can

investigate techniques such as spray drying which is widely available in the food industry and

easy to scale up. It is important to ensure that resveratrol is incorporated into food grade

matrices as the intent is to incorporate the stabilized resveratrol into food products. Protein may

be a desirable encapsulation material for resveratrol due to the binding of resveratrol and

protein which can further enhance stability. The relatively low cost of protein also makes

sodium caseinate a desirable encapsulation material. The sensory properties of resveratrol are

not fully understood. Therefore, it would also be valuable if future research utilized sensory

testing such as consumer testing to evaluate overall acceptance of products with added

resveratrol. Descriptive analysis could also be utilized to evaluate the effect of resveratrol on

attribute intensity of food products.

Overall, future research should focus on innovative processing approaches to increase

resveratrol stability and minimize negative sensory properties of the compound. Thereby,

providing bioactive resveratrol to the consumer and increasing consumer acceptance of

products with added resveratrol.

.

22

2.15 Figures and Tables

Table 2.1: Research supporting the health benefits of resveratrol (bw=body weight)

In-vitro

Author Oral Dosage Cell Model Findings

Bertelli et al [23] 3.56 mg/L Platelet rich plasma Lowered platelet aggregation

Wang et al [24] 10-1000 mM Isolated human platelets

Inhibited platelet aggregation in concentration dependent manner

Li et al [118] 30 µM Endothelial cells Stimulated KCa channels in endothelial cells

Subbaramaiah et al [32]

30 µM Human mammary epithelial cells

70% decrease in basal production of PGE2, inhibited PMA-mediated activation of protein kinase C and COX-2

Schneider et al [33]

25 µM CaCo-2 cells 70% growth inhibition, decreased ornithine decarboxylase activity

Ferry-Dumazet et al [34]

100 mM Normal and leukemic hematopoietic cells

Inhibited proliferation and induced apoptosis of cells

Opipari et al [31] 50-200 µM Human ovarian carcinoma cell lines

Induced cell death in cancer cells separate from apoptosis

Cao and Li [119] 50-100 µM H9C2 cells Resveratrol pretreatment increased protection against cytotoxicity

Mice/Rats

Author Oral Dosage Animal Model Findings

Sharma et al [48]

5, 15, and 50 mg/kg bw/day for 4 weeks

Obese mice Antihyperglycemic activity and improvement in insulin levels

Palsamy et al [49]

5 mg/kg bw/day for 30 days

Diabetic rats Decreased blood glucose, improvements in plasma insulin and hemoglobin

Andersen et al [53]

300 mg/kg bw/day for 8 weeks

Healthy male rats Improved insulin sensitivity

Su et al [52] 0.1-0.75 mg/kg bw

Streptozotocin-induced diabetic mice

Decreased insulin secretion, delayed onset of insulin resistance, increased glucose uptake

Palsamy and Subramanian [50]

5 mg/kg bw/day Diabetic rats Decreased hyperglycemia and increased insulin secretion

23

Barger et al [120]

4.9 mg/kg bw/day for 16 months

Mice on high calorie diet

Prevented decline of cardiac function with age and prevented 93% of age-induced gene expression- similar to results seen with calorie restriction

Pearson et al [25]

5-200 mg/kg bw/day for 6 months

Mice Protected against vascular or kidney dysfunction, reduced total plasma cholesterol and amount of cholesterol carried in lipoprotein fractions

Bradamante et al [26]

25 mg/L for 15 days

Rats Increased recovery at reperfusion and significant vasodilation

Hudersen et al [36]

45µg/kg bw/day for 60 days

Mice Inhibited colon tumorigenesis

Bishayee et al [37]

50, 100, and 200 mg/kg bw/ day

Rats with liver cancer Reduced size of cancer nodules by inhibiting cell proliferation

Jang et al [38] 1-25µM, twice a week for 18 weeks

Mouse skin cancer model

Inhibited development of preneoplastic lesions and inhibit tumorigenesis

Miura et al [1] 50 ppm for 20 days

Hepatoma-bearing rats Solid tumor growth and metastasis was suppressed, serum triglyceride and VLDL + LDL-cholesterol levels suppressed

Kimura et al [39] 2.5 and 10 mg/kg/bw

Lung carcinoma-bearing mice

Reduced tumor volume and weight and lung metastasis

Chen et al [40] 40 mg/kg for 28 days

Mice with neuroblastoma tumors

Suppressed growth rate of subcutaneous neuroblastomas

Brakenhielm et al [41]

1.2 µg/day Mice Inhibited murine fibrosarcoma growth

Revel et al [42] 50 mg/kg/week Balb-C mice Prevented BaP-induced CYP1A1 expression, related to prevention of lung cancer

Schneider et al [43]

0.3-0.4 mg/mouse

C57BL/6J-ApcMin male mice

Prevented colon tumor formation and reduced formation of small intestine tumors by 70%

Garvin et al [44] 100 µM MDA-MB-231 tumors in nude mice

Lowered tumor growth, decreased angiogenesis, increased apoptotic index

Zhou et al [45] 500, 1000, 1500 mg/kg

Gastric cancer cells in nude mice

Induced apoptosis of transplanted tumor cells

Tseng et al [46] 40 mg/kg/day Rats Slower tumor growth, increased survival of animal

Table 2.1 (cont.)

24

Bishayee and Dhir [121]

50, 100, 300 mg/kg bw/day

Sprague-Dawley rats Decreased incidence, number and multiplicity of visible hepatocyte nodules

Kim et al [2] 50-500 nM Mouse model of Alzheimer’s disease

Decreased neurodegeneration and prevent learning impairment

Wang et al [55] 10-8, 10-7, 10-6 g/kg, IV

Cerebral artery occlusion model in Wistar rats

Neuroprotective effect and NF-kappaB p65 expression

Karuppagounder et al [54]

300 mg/kg bw/day for 45 days

Mice Diminished plague formation, brain glutathione decreased and cysteine increased

Gupta et al [57] 20 and 40 mg/kg ip

Male Wistar rats Malondialdehyde levels reduced showing protective effect against seizures

Gupta et al [56] 20 and 40 mg/kg ip

Male Wistar rats Reduced incidence of convulsions, increased malondialdehyde

Baur et al [51] 22.4 mg/kg bw/day

Middle aged mice on high calorie diet

Prevent harmful effects of high calorie intake, increased survival of rats on high calorie diet, increased insulin sensitivity, AMPK, mitochondrial number and improved motor function

Martin et al [122] 5-10 mg/kg/day Rats Reduced degree of colonic injury, the index of neutrophil infiltration and levels of the cytokine, decreased PG2 and COX-2 expression

Lagouge et al [64]

200 and 400 mg/kg bw/day for 15 weeks

Mice Increased aerobic capacity, consumption of oxygen in muscle fibers, protected against diet-induced obesity and insulin resistance

Mizutani et al [123]

1 mg/kg bw/day, gastric intubation

Stroke prone spontaneously hypertensive rats

Suppressed oxidative DNA damage and glycoxidative stress

Wu et al [124] 25, 50, 100 mg/kg, intraperitoneal

Wistar rats after liver transplant

Immuno-suppressive property and protective effect on hepatocytes

Feng et al [125] 0.75-6 µM Mice Promoted lymphocyte proliferation and IL-2 production

Table 2.1 (cont.)

25

Liu et al [126] 2 mg/kg bw Weanling mice Shorten vaginal opening latency periods, enhanced keratinization of vaginal epithelium

Giovannini et al [127]

0.23 µg/kg Male Wistar rats Reduced mortality of ischemic rats and reduced renal damage, inhibited renal peroxidation

Gentilli et al [128]

2 mg/kg Rats Reversed hyperalgesia for 48 hours

Docherty et al [129]

6.25 and 12.5% cream

Mice Delayed or abolished development of herpes

Guinea Pigs


Floreani et al [27]

14 mg/kg for 16 days

Guinea Pigs Increased cardiac DT-diaphorase and catalase activity

Naderali et al [28]

5-70 µM/L Female guinea pigs Concentration dependent relaxation of preconstricted mesenteric and uterine arteries

Rabbits


Wang et al [24] 4 mg/kg bw/day Hypercholesterolemic rabbits

Inhibited ADP-induced platelet aggregation

Wang et al [29] 3 mg/kg bw/day Hypercholesterolemic rabbits

Cardioprotective properties, suppressed atherosclerosis

Elmali et al [130] 10 µM/kg for 2 weeks, IV

Rabbits Decreased cartilage tissue destruction

Humans


Wang et al [24] 10-1000 µM Normotensive males Inhibited platelet aggregation in concentration dependent manner

Kennedy et al [30]

250 and 500 mg dose

Healthy adults Dose dependent increase in cerebral blood flow and deoxyhemoglobin

Wong et al [3] 30, 90, 270 mg Overweight/obese men or post-menopausal women

Improved flow-mediated dilation

Brasnyo et al [4] 10 mg/day for 4 weeks

Type 2 diabetic patients

Reduced insulin resistance and urinary ortho-tyrosine excretion

Brown et al [47] 0.5, 1, 2.5, 5 g/ day for 29 days

Healthy adults Decrease IGF-1 and IGFBP-3 indicating chemoprotection

Table 2.1 (cont.)

26

Timmers et al [5] 150 mg/day for 30 days

Healthy, obese men Reduced sleeping and resting metabolic rate, increased SIRT1 and PDC-1 protein levels, decreased intrahepatic lipid content, circulating glucose, triglycerides and systolic blood pressure

Ghanim et al [6] 40 mg/day for 6 weeks

Healthy, normal weight Reduced reactive, oxygen species and inflammation

Table 2.1 (cont.)

27

2.16 References



3. Wong, R., et al., Acute resveratrol supplementation improves flow-mediated dilatation in overweight/obese individuals with mildly elevated blood pressure. Nutrition, Metabolism and Cardiovascular Diseases, 2011. 21(11): p. 851-856.


5. Timmers, S., et al., Calorie restriction-like effects of 30 days of resveratrol supplementation on energy metabolism and metabolic profile in obese humans. Cell metabolism, 2011. 14(5): p. 612-622.

6. Ghanim, H., et al., An antiinflammatory and reactive oxygen species suppressive effects of an extract of polygonum cuspidatum containing resveratrol. Journal of Clinical Endocrinology & Metabolism, 2010. 95(9): p. E1-E8.

7. Filip, V., et al., Resveratrol and its antioxidant and antimicrobial effectiveness. Food chemistry, 2003. 83(4): p. 585-593.

8. Dani, C., et al., Antioxidant protection of resveratrol and catechin in saccharomyces cerevisiae. Journal of Agricultural and Food Chemistry, 2008. 56(11): p. 4268-4272.

9. Trela, B.C. and A.L. Waterhouse, Resveratrol: Isomeric molar absorptivities and stability. Journal of Agricultural and Food Chemistry, 1996. 44(5): p. 1253-1257.

10. Aldrich, S., Resveratrol sigma prod. No. R5010. 1997.

11. Wenzel, E. and V. Somoza, Metabolism and bioavailability of trans‐resveratrol. Molecular nutrition & food research, 2005. 49(5): p. 472-481.


13. Lucas-Abellán, C., et al., Cyclodextrins as resveratrol carrier system. Food Chemistry, 2007. 104(1): p. 39-44.

14. Nam, J.B., et al., Stabilization of resveratrol immobilized in monodisperse cyano-functionalized porous polymeric microspheres. Polymer, 2005. 46(21): p. 8956-8963.

15. Peng, H., et al., Vanillin cross-linked chitosan microspheres for controlled release of resveratrol. Food chemistry, 2010. 121(1): p. 23-28.

16. Das, S. and K.Y. Ng, Colon-specific delivery of resveratrol: Optimization of multi-particulate calcium-pectinate carrier. International Journal of Pharmaceutics, 2010. 385(1-2): p. 20-28.

17. Hemar, Y., et al., Encapsulation of resveratrol using water-in-oil-in-water double emulsions. Food Biophysics, 2010. 5(2): p. 120-127.

18. Sessa, M., et al., Evaluation of the stability and antioxidant activity of nanoencapsulated resveratrol during in vitro digestion. Journal of Agricultural and Food Chemistry, 2011.

19. Pando, D., et al., Preparation and characterization of niosomes containing resveratrol. Journal of Food Engineering, 2013.

20. Wan, Z.-L., et al., Enhanced physical and oxidative stability of soy protein-based emulsions by the incorporation of water-soluble stevioside-resevratrol complex. Journal of agricultural and food chemistry, 2013.

28

21. Spigno, G., et al., Nanoencapsulation systems to improve solubility and antioxidant efficiency of a grape marc extract into hazelnut paste. Journal of Food Engineering, 2012.

22. Pastor, C., et al., Physical and antioxidant properties of chitosan and methylcellulose based films containing resveratrol. Food Hydrocolloids, 2012.

23. Bertelli, A., et al., Antiplatelet activity of synthetic and natural resveratrol in red wine. International journal of tissue reactions, 1995. 17(1): p. 1.


25. Pearson, K.J., et al., Resveratrol delays age-related deterioration and mimics transcriptional aspects of dietary restriction without extending life span. Cell metabolism, 2008. 8(2): p. 157-168.

26. Bradamante, S., et al., Resveratrol provides late-phase cardioprotection by means of a nitric oxide-and adenosine-mediated mechanism. European journal of pharmacology, 2003. 465(1): p. 115-123.

27. Floreani, M., et al., Oral administration of trans-resveratrol to guinea pigs increases cardiac dt-diaphorase and catalase activities, and protects isolated atria from menadione toxicity. Life sciences, 2003. 72(24): p. 2741-2750.

28. Naderali, E.K., P.J. Doyle, and G. Williams, Resveratrol induces vasorelaxation of mesenteric and uterine arteries from female guinea-pigs. Clinical Science, 2000. 98(5): p. 537-543.

29. Wang, Z., et al., Dealcoholized red wine containing known amounts of resveratrol suppresses atherosclerosis in hypercholesterolemic rabbits without affecting plasma lipid levels. International Journal of Molecular Medicine, 2005. 16(4): p. 533.

30. Kennedy, D.O., et al., Effects of resveratrol on cerebral blood flow variables and cognitive performance in humans: A double-blind, placebo-controlled, crossover investigation. The American journal of clinical nutrition, 2010. 91(6): p. 1590-1597.

31. Opipari, A.W., et al., Resveratrol-induced autophagocytosis in ovarian cancer cells. Cancer Research, 2004. 64(2): p. 696.

32. Subbaramaiah, K., et al., Resveratrol inhibits cyclooxygenase-2 transcription and activity in phorbol ester-treated human mammary epithelial cells. Journal of Biological Chemistry, 1998. 273(34): p. 21875-21882.

33. Schneider, Y., et al., Anti-proliferative effect of resveratrol, a natural component of grapes and wine, on human colonic cancer cells. Cancer letters, 2000. 158(1): p. 85-91.

34. Ferry-Dumazet, H., et al., Resveratrol inhibits the growth and induces the apoptosis of both normal and leukemic hematopoietic cells. Carcinogenesis, 2002. 23(8): p. 1327-1333.

35. Baur, J.A. and D.A. Sinclair, Therapeutic potential of resveratrol: The in vivo evidence. Nature Reviews Drug Discovery, 2006. 5(6): p. 493-506.

36. Huderson, A.C., et al., Chemoprevention of benzo (a) pyrene-induced colon polyps in apc min mice by resveratrol. The Journal of Nutritional Biochemistry, 2012.

37. Bishayee, A. and N. Dhir, Resveratrol-mediated chemoprevention of diethylnitrosamine-initiated hepatocarcinogenesis: Inhibition of cell proliferation and induction of apoptosis. Chemico-biological interactions, 2009. 179(2-3): p. 131-144.

38. Jang, M., et al., Cancer chemopreventive activity of resveratrol, a natural product derived from grapes. Science, 1997. 275(5297): p. 218-220.

39. Kimura, Y. and H. Okuda, Resveratrol isolated from polygonum cuspidatum root prevents tumor growth and metastasis to lung and tumor-induced neovascularization in lewis lung carcinoma-bearing mice. The Journal of nutrition, 2001. 131(6): p. 1844-1849.

29

40. Chen, Y., et al., Resveratrol-induced cellular apoptosis and cell cycle arrest in neuroblastoma cells and antitumor effects on neuroblastoma in mice. Surgery, 2004. 136(1): p. 57-66.

41. Bråkenhielm, E., R. Cao, and Y. Cao, Suppression of angiogenesis, tumor growth, and wound healing by resveratrol, a natural compound in red wine and grapes. The FASEB Journal, 2001. 15(10): p. 1798-1800.

42. Revel, A., et al., Resveratrol, a natural aryl hydrocarbon receptor antagonist, protects lung from DNA damage and apoptosis caused by benzo [a] pyrene. Journal of Applied Toxicology, 2003. 23(4): p. 255-261.

43. Schneider, Y., et al., Resveratrol inhibits intestinal tumorigenesis and modulates host-defense-related gene expression in an animal model of human familial adenomatous polyposis. Nutrition and cancer, 2001. 39(1): p. 102-107.

44. Garvin, S., K. Öllinger, and C. Dabrosin, Resveratrol induces apoptosis and inhibits angiogenesis in human breast cancer xenografts in vivo. Cancer letters, 2006. 231(1): p. 113-122.

45. Zhou, H.B., et al., Anticancer activity of resveratrol on implanted human primary gastric carcinoma cells in nude mice. World J Gastroenterol, 2005. 11(2): p. 280-4.

46. Tseng, S.H., et al., Resveratrol suppresses the angiogenesis and tumor growth of gliomas in rats. Clinical Cancer Research, 2004. 10(6): p. 2190-2202.

47. Brown, V.A., et al., Repeat dose study of the cancer chemopreventive agent resveratrol in healthy volunteers: Safety, pharmacokinetics, and effect on the insulin-like growth factor axis. Cancer Research, 2010. 70(22): p. 9003-9011.

48. Sharma, S., et al., Antidiabetic activity of resveratrol, a known sirt1 activator in a genetic model for type‐2 diabetes. Phytotherapy Research, 2011. 25(1): p. 67-73.

49. Palsamy, P. and S. Subramanian, Resveratrol, a natural phytoalexin, normalizes hyperglycemia in streptozotocin-nicotinamide induced experimental diabetic rats. Biomedicine & Pharmacotherapy, 2008. 62(9): p. 598-605.

50. Palsamy, P. and S. Subramanian, Ameliorative potential of resveratrol on proinflammatory cytokines, hyperglycemia mediated oxidative stress, and pancreatic β‐cell dysfunction in streptozotocin‐nicotinamide‐induced diabetic rats. Journal of Cellular Physiology, 2010. 224(2): p. 423-432.

51. Baur, J.A., et al., Resveratrol improves health and survival of mice on a high-calorie diet. Nature, 2006. 444(7117): p. 337-342.

52. Su, H.C., L.M. Hung, and J.K. Chen, Resveratrol, a red wine antioxidant, possesses an insulin-like effect in streptozotocin-induced diabetic rats. American Journal of Physiology-Endocrinology And Metabolism, 2006. 290(6): p. E1339.

53. Andersen, G., et al., High dose of dietary resveratrol enhances insulin sensitivity in healthy rats but does not lead to metabolite concentrations effective for sirt1 expression. Molecular nutrition & food research, 2011.

54. Karuppagounder, S.S., et al., Dietary supplementation with resveratrol reduces plaque pathology in a transgenic model of alzheimer's disease. Neurochemistry international, 2009. 54(2): p. 111-118.

55. Wang, Y., F. He, and X. Li, [the neuroprotection of resveratrol in the experimental cerebral ischemia]. Zhonghua Yi Xue Za Zhi, 2003. 83(7): p. 534.

56. Gupta, Y., S. Briyal, and G. Chaudhary, Protective effect of trans-resveratrol against kainic acid-induced seizures and oxidative stress in rats. Pharmacology Biochemistry and Behavior, 2002. 71(1): p. 245-249.

57. Gupta, Y., et al., Protective effect of resveratrol against intracortical fecl3-induced model of posttraumatic seizures in rats. Methods Find Exp Clin Pharmacol, 2001. 23(5): p. 241-244.

30

58. Chen, J., et al., Sirt1 protects against microglia-dependent amyloid-β toxicity through inhibiting nf-κb signaling. Journal of Biological Chemistry, 2005. 280(48): p. 40364-40374.

59. Howitz, K.T., et al., Small molecule activators of sirtuins extend saccharomyces cerevisiae lifespan. Nature, 2003. 425(6954): p. 191-196.

60. Wood, J.G., et al., Sirtuin activators mimic caloric restriction and delay ageing in metazoans. Nature, 2004. 430(7000): p. 686-689.

61. Bauer, J.H., et al., An accelerated assay for the identification of lifespan-extending interventions in drosophila melanogaster. Proceedings of the National Academy of Sciences of the United States of America, 2004. 101(35): p. 12980.

62. Weindruch, R., Caloric restriction and aging. Scientific American, 1996. 274(1): p. 46. 63. Valenzano, D.R., et al., Resveratrol prolongs lifespan and retards the onset of age-

related markers in a short-lived vertebrate. Current Biology, 2006. 16(3): p. 296-300. 64. Lagouge, M., et al., Resveratrol improves mitochondrial function and protects against

metabolic disease by activating sirt1 and pgc-1 [alpha]. Cell, 2006. 127(6): p. 1109-1122. 65. Kim, S., et al., Resveratrol exerts anti-obesity effects via mechanisms involving down-

regulation of adipogenic and inflammatory processes in mice. Biochemical pharmacology, 2011. 81(11): p. 1343-1351.

66. Dal-Pan, A., S. Blanc, and F. Aujard, Resveratrol suppresses body mass gain in a seasonal non-human primate model of obesity. BMC physiology, 2010. 10(1): p. 11.

67. Heredia, A., et al., Targeting host nucleotide biosynthesis with resveratrol inhibits emtricitabine-resistant hiv-1. AIDS, 2014. 28(3): p. 317-323.

68. Zhang, H.-S., et al., Resveratrol inhibited tat-induced hiv-1 ltr transactivation via nad+-dependent sirt1 activity. Life sciences, 2009. 85(13): p. 484-489.

69. Burns, J., et al., Plant foods and herbal sources of resveratrol. Journal of agricultural and food chemistry, 2002. 50(11): p. 3337-3340.

70. Lyons, M.M., et al., Resveratrol in raw and baked blueberries and bilberries. Journal of agricultural and food chemistry, 2003. 51(20): p. 5867-5870.

71. Sanders, T.H., R.W. McMichael Jr, and K.W. Hendrix, Occurrence of resveratrol in edible peanuts. Journal of Agricultural and Food Chemistry, 2000. 48(4): p. 1243-1246.

72. Siemann, E. and L. Creasy, Concentration of the phytoalexin resveratrol in wine. American Journal of Enology and Viticulture, 1992. 43(1): p. 49-52.

73. Tang, K., et al., Changes of resveratrol and antioxidant enzymes during uv-induced plant defense response in peanut seedlings. Journal of plant physiology, 2010. 167(2): p. 95-102.

74. Wang, W., et al., Distribution of resveratrol and stilbene synthase in young grape plants ( vitis vinifera l. Cv. Cabernet sauvignon) and the effect of uv-c on its accumulation. Plant Physiology and Biochemistry, 2010. 48(2): p. 142-152.

75. Adrian, M., et al., Induction of phytoalexin (resveratrol) synthesis in grapevine leaves treated with aluminum chloride (alcl3). Journal of agricultural and food chemistry, 1996. 44(8): p. 1979-1981.

76. Jeandet, P., et al., Production of the phytoalexin resveratrol by grapes as a response to botrytis attack under natural conditions. Journal of Phytopathology, 1995. 143(3): p. 135-139.

77. Langcake, P., Disease resistance of vitis spp. And the production of the stress metabolites resveratrol, ε-viniferin, α-viniferin and pterostilbene. Physiological Plant Pathology, 1981. 18(2): p. 213-226.

78. Liu, S., et al., High content of resveratrol in lettuce transformed with a stilbene synthase gene of parthenocissus henryana. Journal of agricultural and food chemistry, 2006. 54(21): p. 8082-8085.

31

79. Rühmann, S., et al., Piceid (resveratrol glucoside) synthesis in stilbene synthase transgenic apple fruit. Journal of agricultural and food chemistry, 2006. 54(13): p. 4633-4640.

80. Jensen, J.S., C.F. Wertz, and V.A. O’Neill, Preformulation stability of trans-resveratrol and trans-resveratrol glucoside (piceid). Journal of Agricultural and Food Chemistry, 2010. 58(3): p. 1685-1690.

81. Food, U., Drug administration. Guidance for industry botanical drug products. 2004. 82. Food, U., Drug administration. Dietary supplement health and education act of 1994.

Public law, 2006. 103: p. 417. 83. Williams, L.D., et al., Safety studies conducted on high-purity trans-resveratrol in

experimental animals. Food and Chemical Toxicology, 2009. 47(9): p. 2170-2182. 84. Boocock, D.J., et al., Phase i dose escalation pharmacokinetic study in healthy

volunteers of resveratrol, a potential cancer chemopreventive agent. Cancer Epidemiology Biomarkers & Prevention, 2007. 16(6): p. 1246-1252.

85. Almeida, L., et al., Pharmacokinetic and safety profile of trans‐resveratrol in a rising multiple‐dose study in healthy volunteers. Molecular nutrition & food research, 2009. 53(S1): p. S7-S15.

86. Figueiras, T.S., M.T. Neves-Petersen, and S.B. Petersen, Activation energy of light induced isomerization of resveratrol. Journal of fluorescence, 2011. 21(5): p. 1897-1906.


88. Allan, K.E., C.E. Lenehan, and A.V. Ellis, Uv light stability of α-cyclodextrin/resveratrol host–guest complexes and isomer stability at varying ph. Australian journal of chemistry, 2009. 62(8): p. 921-926.

89. Hung, C.F., et al., Development and evaluation of emulsion-liposome blends for resveratrol delivery. Journal of Nanoscience and Nanotechnology, 6, 2006. 9(10): p. 2950-2958.

90. Shi, G., et al., Stabilization and encapsulation of photosensitive resveratrol within yeast cell. International Journal of Pharmaceutics, 2008. 349(1): p. 83-93.

91. Goldberg, D.M., J. Yan, and G.J. Soleas, Absorption of three wine-related polyphenols in three different matrices by healthy subjects. Clinical biochemistry, 2003. 36(1): p. 79-87.


93. Azorín Ortuño, M., et al., Metabolites and tissue distribution of resveratrol in the pig. Molecular nutrition & food research.

94. Mantegna, S., et al., A one-pot ultrasound-assisted water extraction/cyclodextrin encapsulation of resveratrol from polygonum cuspidatum. Food Chemistry, 2012. 130(3): p. 746-750.

95. Lu, Z., et al., Complexation of resveratrol with cyclodextrins: Solubility and antioxidant activity. Food Chemistry, 2009. 113(1): p. 17-20.

96. Lucas-Abellán, C., et al., Orac-fluorescein assay to determine the oxygen radical absorbance capacity of resveratrol complexed in cyclodextrins. Journal of agricultural and food chemistry, 2008. 56(6): p. 2254-2259.

97. Lu, Z., et al., Cytotoxicity and inhibition of lipid peroxidation activity of resveratrol/cyclodextrin inclusion complexes. Journal of Inclusion Phenomena and Macrocyclic Chemistry, 2012. 73(1-4): p. 313-320.

98. Mourtzinos, I., et al., Thermal oxidation of vanillin affects its antioxidant and antimicrobial properties. Food Chemistry, 2009. 114(3): p. 791-797.

32

99. Sinha, A.K., U.K. Sharma, and N. Sharma, A comprehensive review on vanilla flavor: Extraction, isolation and quantification of vanillin and others constituents. International journal of food sciences and nutrition, 2008. 59(4): p. 299-326.

100. Das, S. and K.Y. Ng, Resveratrol‐loaded calcium‐pectinate beads: Effects of formulation parameters on drug release and bead characteristics. Journal of pharmaceutical sciences, 2010. 99(2): p. 840-860.

101. Das, S., K.Y. Ng, and P.C. Ho, Formulation and optimization of zinc-pectinate beads for the controlled delivery of resveratrol. Aaps Pharmscitech, 2010. 11(2): p. 729-742.

102. Gokce, E.H., et al., Resveratrol-loaded solid lipid nanoparticles versus nanostructured lipid carriers: Evaluation of antioxidant potential for dermal applications. International journal of nanomedicine, 2012. 7: p. 1841.

103. Caddeo, C., et al., Effect of resveratrol incorporated in liposomes on proliferation and uv-b protection of cells. International journal of pharmaceutics, 2008. 363(1): p. 183-191.

104. Ahn, S.-I., Y.-K. Lee, and H.-S. Kwak, Optimization of water-in-oil-in-water microencapsulated β-galactosidase by response surface methodology. Journal of microencapsulation, 2013. 30(5): p. 460-469.

105. Lee, C.-W., et al., Resveratrol nanoparticle system improves dissolution properties and enhances the hepatoprotective effect of resveratrol through antioxidant and anti-inflammatory pathways. Journal of agricultural and food chemistry, 2012. 60(18): p. 4662-4671.

106. Augustin, M., et al., Effects of microencapsulation on the gastrointestinal transit and tissue distribution of a bioactive mixture of fish oil, tributyrin and resveratrol. Journal of Functional Foods, 2011. 3(1): p. 25-37.

107. Liang, L., H. Tajmir-Riahi, and M. Subirade, Interaction of β-lactoglobulin with resveratrol and its biological implications. Biomacromolecules, 2007. 9(1): p. 50-56.

108. Liang, L. and M. Subirade, Study of the acid and thermal stability of β-lactoglobulin–ligand complexes using fluorescence quenching. Food Chemistry, 2012. 132(4): p. 2023-2029.

109. Acharya, D.P., L. Sanguansri, and M.A. Augustin, Binding of resveratrol with sodium caseinate in aqueous solutions. Food chemistry, 2013. 141(2): p. 1050-1054.

110. Hemar, Y., et al., Investigation into the interaction between resveratrol and whey proteins using fluorescence spectroscopy. International Journal of Food Science & Technology, 2011. 46(10): p. 2137-2144.

111. Isailović, B.D., et al., Resveratrol loaded liposomes produced by different techniques. Innovative Food Science & Emerging Technologies, 2013. 19: p. 181-189.

112. Shao, J., et al., Enhanced growth inhibition effect of resveratrol incorporated into biodegradable nanoparticles against glioma cells is mediated by the induction of intracellular reactive oxygen species levels. Colloids and Surfaces B: Biointerfaces, 2009. 72(1): p. 40-47.

113. Neves, A.R., et al., Novel resveratrol nanodelivery systems based on lipid nanoparticles to enhance its oral bioavailability. International journal of nanomedicine, 2013. 8: p. 177.

114. Spigno, G., et al., Nanoencapsulation systems to improve solubility and antioxidant efficiency of a grape marc extract into hazelnut paste. Journal of Food Engineering, 2013. 114(2): p. 207-214.

115. Hwang, S.W., et al., Poly (l‐lactic acid) with added α‐tocopherol and resveratrol: Optical, physical, thermal and mechanical properties. Polymer International, 2012. 61(3): p. 418-425.

116. Hwang, S.W., et al., Migration of α-tocopherol and resveratrol from poly (l-lactic acid)/starch blends films into ethanol. Journal of Food Engineering, 2013.

33

117. Lante, A., et al., Chemical parameters, biologically active polyphenols and sensory characteristics of some italian organic wines. Journal of Wine Research, 2004. 15(3): p. 203-209.

118. Li, H.F., S.A. Chen, and S.N. Wu, Evidence for the stimulatory effect of resveratrol on ca2+-activated k+ current in vascular endothelial cells. Cardiovascular research, 2000. 45(4): p. 1035-1045.

119. Cao, Z. and Y. Li, Potent induction of cellular antioxidants and phase 2 enzymes by resveratrol in cardiomyocytes: Protection against oxidative and electrophilic injury. European journal of pharmacology, 2004. 489(1-2): p. 39.

120. Barger, J.L., et al., A low dose of dietary resveratrol partially mimics caloric restriction and retards aging parameters in mice. PLoS One, 2008. 3(6): p. e2264.

121. Bishayee, A. and N. Dhir, Resveratrol-mediated chemoprevention of diethylnitrosamine-initiated hepatocarcinogenesis: Inhibition of cell proliferation and induction of apoptosis. Chemico-biological interactions, 2009. 179(2): p. 131-144.

122. Martı́n, A.R., et al., Resveratrol, a polyphenol found in grapes, suppresses oxidative damage and stimulates apoptosis during early colonic inflammation in rats. Biochemical pharmacology, 2004. 67(7): p. 1399-1410.

123. Mizutani, K., et al., Protective effect of resveratrol on oxidative damage in male and female stroke‐prone spontaneously hypertensive rats. Clinical and Experimental Pharmacology and Physiology, 2003. 28(1‐2): p. 55-59.

124. Wu, S.L., et al., Resveratrol prolongs allograft survival after liver transplantation in rats. WORLD JOURNAL OF GASTROENTEROLOGY, 2005. 11(30): p. 4745.

125. Feng, Y.H., et al., Low dose of resveratrol enhanced immune response of mice. Acta Pharmacologica Sinica, 2002. 23(10): p. 893-897.

126. Liu, Z., et al., [estrogenicity of trans-resveratrol in immature mice in vivo]. Wei sheng yan jiu= Journal of hygiene research, 2002. 31(3): p. 188.

127. Giovannini, L., et al., Resveratrol, a polyphenol found in wine, reduces ischemia reperfusion injury in rat kidneys. Journal of cardiovascular pharmacology, 2001. 37(3): p. 262.

128. Gentilli, M., et al., Resveratrol decreases hyperalgesia induced by carrageenan in the rat hind paw. Life sciences, 2001. 68(11): p. 1317-1321.

129. Docherty, J.J., et al., Effect of resveratrol on herpes simplex virus vaginal infection in the mouse. Antiviral research, 2005. 67(3): p. 155-162.

130. Elmali, N., et al., Effect of resveratrol in experimental osteoarthritis in rabbits. Inflammation research, 2005. 54(4): p. 158-162.

34

CHAPTER 3: STABILITY AND BINDING OF TRANS-RESVERATROL ENCAPSULATED IN A PROTEIN MATRIX PRODUCED USING SPRAY DRYING

3.1 Abstract

Resveratrol has demonstrated the potential to provide therapeutic and preventive

activities against diseases such as heart disease and cancer. The incorporation of resveratrol

into food products would allow for wide access of these health benefits to a larger population,

but this strategy is limited by instability of resveratrol under environmental conditions and within

the digestive system due to the isomerization of trans-resveratrol (bioactive form) to cis-

resveratrol (bio-inactive form). The overall goal of this research was to stabilize the bioactive

form of resveratrol through an innovative processing approach, microencapsulation. Trans-

resveratrol was encapsulated using whey protein concentrate or sodium caseinate, with or

without anhydrous milk fat (AMF). The binding of protein and resveratrol is of interest because

it may help to stabilize resveratrol. Resveratrol-protein binding was calculated utilizing the

Stern-Volmer equation and the developed resveratrol binding sites equation. The stability of

encapsulated resveratrol was evaluated after exposure to Ultraviolet A (UVA) light and in-vitro

digestion. The samples were exposed to UVA light, equivalent to about 4 min of sunlight, in

aqueous solution. Digestive stability was assessed using a 3-phase in-vitro digestion, which

included oral, gastric and small intestine phases. Resveratrol isomers were quantified by

reverse phase HPLC with coulometric detection. After UVA light exposure, sodium-caseinate-

based microcapsules retained a significantly higher trans:cis resveratrol ratio (0.63) than whey-

protein-concentrate-based microcapsules (0.43) and unencapsulated resveratrol (0.49). In

addition, all encapsulated resveratrol had an increased digestive stability and bioaccessibility,

respectively, in comparison to unencapsulated resveratrol (47% and 23%), with sodium

caseinate providing a higher digestive stability (84% and 60%) compared to whey-protein-

concentrate-based microcapsules (70% and 53%). The addition of AMF within formulations did

not affect UVA light and in-vitro digestion stability. In conclusion, microencapsulation with

35

sodium caseinate increased the stability of resveratrol after UVA light exposure and in-vitro

digestion conditions. This encapsulation-system-approach can be extended to other labile,

bioactive polyphenols.

Keywords: resveratrol, light stability, bioaccessibility, binding, microencapsulation

3.2 Introduction

Resveratrol is a polyphenol found in grapes and peanuts that has gained interest due to

the numerous health benefits associated with this compound. Resveratrol naturally occurs in

low amount in red grapes (0.050 mg/100 g), red wine (0.002-0.653 mg/L) and peanuts (0.002-

.0179 mg/100 g) [1-3]. The incorporation of resveratrol into food products would allow the

intake of biologically active dosages of resveratrol and disseminate its health benefits to a larger

population. The health benefits of resveratrol have been extensively investigated over the years

in a wide range of in-vitro, pre-clinical and clinical settings. In-vitro studies showed that

resveratrol can lower platelet aggregation thereby decreasing cardiovascular disease and

promoting cell death in cancer cells [4, 5]. In mice and rat models, resveratrol has been shown

to decrease blood glucose related to diabetes and reduce plasma cholesterol linked to a

reduction in risk of cardiovascular disease [6, 7]. Resveratrol has also been shown to decrease

tumor growth and neurodegeneration in mice and rat models [8, 9]. In humans, several

randomized control trials have shown the effects of resveratrol on improvement of cerebral

blood flow and flow mediated dilation which are related to decreased risk of stroke, reduced

insulin resistance, modulation of biochemical pathways associated with chemoprotection and

inflammation [10-14].

The preferred dietary form of resveratrol which has been associated with benefits is

trans-resveratrol while the form which has questionable health benefits is cis-resveratrol. The

activation energy to cause the isomerization from the trans to cis-isomer was about 3.7 kcal/M

which is a relatively small amount of energy in comparison to the activation energy to convert

36

hydrogen peroxide to oxygen, which was about 10 kcal/M [15, 16]. The conversion from the

bioactive to bio-inactive form of resveratrol is induced by environmental conditions such as light,

heat and pH [17-20]. Under UV light, 90.6% of trans-resveratrol in solution was converted to

cis-resveratrol after 120 min exposure at 366 nm [17]. Also, one hour of exposure to sunlight

resulted in 80-90% conversion of trans-resveratrol to cis-resveratrol [18]. Resveratrol is more

stable to heat than light. In blueberries and bilberries, 17-46% of resveratrol was degraded after

heating for 18 min at 190°C [19]. Trans-resveratrol was stable in a range of pH conditions, from

pH 3 to 7 for one month, but unstable at pH 12 with a half-life ranging from 10-20 hr [20].

Another study found that trans-resveratrol was stable between pH 1-7 for 28 days and the half-

life of trans-resveratrol in pH 10 was 1.6 hr [17]. Therefore, prior research showed that

resveratrol is unstable in certain environmental conditions.

Digestive stability and bioaccessibility of resveratrol is another important consideration in

order to ensure bioactivity of the compound when consumed. An oral dose of resveratrol has

been shown to have 70% absorption, but only trace amounts of trans-resveratrol were found in

the blood after consumption of the compound. Resveratrol conjugated with sulfates and

glucuronic acid were the most common metabolites found in the urine and plasma [21]. After

oral ingestion of resveratrol, trace amounts of the ingested resveratrol was detected in plasma

and glucuronides were the most common form of resveratrol found in the urine [22]. Peak

plasma concentration of trans-resveratrol was less than 2% of the oral dose and occurred about

30 min after consumption [23]. Thus, this low digestive stability and bioavailability of resveratrol

provides further rationale to stabilize resveratrol in order to ensure the health benefits are

delivered to the consumer.

Encapsulation is a viable processing method that can help to stabilize the bioactive form

of resveratrol to environmental and digestive stress. This technique is used to incorporate labile

or desirable compounds into microcapsules utilizing a stable wall material. The core ingredient

can be protected from environmental conditions such as light, heat, pH and moisture. In the

37

food industry, encapsulation is common for flavors, enzymes, colors, preservatives,

antioxidants, nutrients and artificial sweeteners [24]. Encapsulation can be accomplished using

several methods which include spray drying, liposomes, coacervation, and emulsions [25]. In

the food industry spray drying is favored because it is economical, flexible, a continuous

operation, and produces particles of high quality [25]. One limitation of spray drying is the wall

material needs to be water soluble to form a solution [26]. Therefore, the selection of wall

materials is limited by their solubility in water. Some wall materials that have been used in

spray drying are maltodextrins, chitosan, gum acacia and protein-lipid mixtures [26, 27]. In a

prior study, porous polymeric microspheres were used to encapsulate resveratrol and the

wetting time and presence of cyano-functional groups were measured. In addition, antioxidant

potential was measured and it was found that encapsulation helped to preserve 93% of the

antioxidant activity and maintain the bioactivity for 5 weeks [28]. These results suggested that

the encapsulation of resveratrol will help to stabilize the compound which in turn may enhance

bioaccessibility. In another study, the incorporation of resveratrol into cyclodextrin complexes

was shown to maintain its antioxidant potential and increase solubility of the compound [29].

Cross-linked chitosan and pectinate beads have also been utilized to encapsulate resveratrol

[30, 31]. Results from these encapsulation studies indicate that protection of resveratrol is

feasible, but requires optimization. The current methods, although useful, are limited for their

wide use in industry, since they are more appropriate for small scale production. Protein such

as sodium caseinate has been used in the past for encapsulation of curcumin, thymol, and a

combination of bioactive compounds [32-34].

In this study, spray drying with protein-based material was used as the encapsulation

method. Sodium caseinate and whey protein concentrate were chosen as wall materials to

encapsulate resveratrol because they are common dairy protein utilized in the food industry and

can be readily leveraged in many product formats, making commercialization more feasible. In

addition, resveratrol has been previously shown to bind with protein thereby stability of the

38

compound would be enhanced [35-37]. The binding of resveratrol to β-lactoglobulin decreased

the isomerization of resveratrol in comparison to resveratrol not bound to protein [38]. Future

research can utilize plant based proteins such as soy protein and pea protein, in order to make

the encapsulation system suitable for vegans or those with sensitivities to dairy.

The objective of this study was to evaluate the stability of the bioactive form of

resveratrol, trans-resveratrol, against light and digestive conditions, after microencapsulation.

The microcapsules were characterized and evaluated on the basis of stability under UVA light

and in-vitro digestion. The hypothesis was that microencapsulation of resveratrol within a

protein matrix will change the stability of resveratrol in comparison to unencapsulated

resveratrol. The second objective of this study was to quantify the amount of binding between

resveratrol and protein utilizing two equations and methods of calculation. The hypothesis was

that protein binding plays a significant role in the stability of resveratrol which can explain the

partial recovery of resveratrol from the microcapsules.

3.3 Materials and Methods

3.3.1 Materials

Trans-resveratrol was supplied by Dutch State Mines (DSM, Parsippany, NJ); its purity

was >99% according to the manufacturer. Oxy-resveratrol was used as an internal standard for

high performance liquid chromatography (HPLC), and it was purchased from Cayman chemicals

(Ann Arbor, MI); its purity was >98%. Whey protein concentrate and sodium caseinate were

supplied by Agropur (La Crosse, WI). Anhydrous milk fat was purchased from Danish maid

(Chicago, IL). Methanol was HPLC grade and purchased from Sigma-Aldrich (St. Louis, MO).

Materials used for the in-vitro digestion study were all purchased from Sigma-Aldrich (St. Louis,

MO). The filters used were 0.2 µM PTFE filter (VWR, Radnor, PA).

39

3.3.2 Methods

3.3.2.1 Microcapsule Production

Resveratrol microcapsules were spray dried in duplicates according to the formulas

displayed in Table 3.1 and a schematic diagram of the microcapsule production process is

shown in Figure 3.1. Whey protein concentrate or sodium caseinate (Agropur Ingredients, La

Crosse, WI) were mixed thoroughly with deionized water and heat treated at 80ºC in a water

bath shaker (C76 Water Bath Shaker, New Brunswick Scientific, Edison, NJ) at 100 rpm for 25

min (whey protein concentrate) or 2 hours (sodium caseinate). The extended period of heat

treatment of sodium caseinate was used to enable even dispersion of the protein within the

solution. Anhydrous milk fat (AMF) and trans-resveratrol were mixed together for 2 min at

15,200 rpm using a hand mixer (IKA Works, Wilmington, NC). Then, the AMF and resveratrol

mixture was added into the protein solution. High-pressure homogenization (APV Gaulin Inc.,

Wilmington, MA) at 55 MPa was, then, used to disperse the mixture evenly and create an

emulsion. The solution was passed through the homogenizer twice. The homogenized sample

was incubated at 45ºC in a water bath for an hour to ensure it reached temperature equilibrium

throughout the solution. The temperature of incubation was chosen because it is above the

melting point of anhydrous milk fat [39].

A spray dryer (B-290 Buchi Corporation, New Castle, DE) was used to produce the

encapsulated resveratrol particles. The spray drying conditions were: inlet temperature =

160ºC, outlet temperature = 90ºC, flow rate = 5-7 g/min, air pressure = 5 kPa and nozzle

diameter = 0.7 mm. Flow rate was adjusted to maintain outlet temperature. Throughout the

process, aluminum foil was used to protect the resveratrol from direct exposure to light.

3.3.2.2 High-performance Liquid Chromatography

The reverse phase high-performance liquid chromatography (HPLC) with coulometric

detection consisted of Waters 717plus autosampler (Milford, MA) and ESA CoulArray detector

(ThermoScientific, Sunnyvale, CA), with a Phenomenex Gemini 5u, C18, 110A, 150x4.6 mm

40

column (Phenomenex, Torrance, CA). A binary mobile phase was used, 100% methanol and

25 mM sodium acetate at pH 4.5 (Sigma, St. Louis, MO). A gradient separation method was

used starting with 30% methanol and held for 2 min. Then, methanol was increased to 60%

during 14 min and held for 3 min. Then, the gradient was brought back to initial conditions over

2 min and held constant for 2 min. Oxy-resveratrol was used as an internal standard in HPLC

analysis. External standard curves were constructed with pure compounds of trans-resveratrol,

cis-resveratrol and oxy-resveratrol.

3.3.2.3 Moisture Content and Water Activity Measurements

Moisture content was analyzed by HR83 Halogen Moisture Analyzer (Mettler Toledo,

Columbus, OH), and water activity was assessed by Aqua Lab 4TE (Aqua Lab Technologies,

Riverside, CA). Samples from each replication of spray drying were measured in triplicates.

3.3.2.4 Morphology and Particle Size

Microcapsule morphology was observed through scanning electron microscope (XL30

ESEM-FEG, FEI Company, Hillsboro, OR) at Beckmann Institute for Advanced Science and

Technology (Urbana, IL). Samples were coated with gold-palladium using a sputter coater

(Desk-1 TSC, Denton Vacuum, Moorestown, NJ). Hivac mode was used to observe the

morphology of the resveratrol microcapsules at a voltage of 5 kV. Particle size was estimated

from the scanning electron microscope images.

3.3.2.5 Microencapsulation Efficiency Measurements

Microencapsulation efficiency (ME) was defined as the fraction of resveratrol that is not

washed off by a solvent. Fifty mg of resveratrol microcapsules and 5 mL methanol were

combined in a flask and placed on a shaker at 100 rpm for 15 min at room temperature. The

solution was centrifuged at 1775 g with a Sorvall Legend Micro 17 centrifuge (ThermoScientific,

Sunnyvale, CA) for 5 min at 23°C with and resveratrol content of the supernatant was measured

by HPLC. Samples were filtered with 0.2 µM PTFE filters before HPLC injection.

Microencapsulation efficiency was calculated using the following equation [40]:

41

ME (%) = (𝒕𝒐𝒕𝒂𝒍 𝒓𝒆𝒔𝒗𝒆𝒓𝒂𝒕𝒓𝒐𝒍 −𝒔𝒖𝒓𝒇𝒂𝒄𝒆 𝒓𝒆𝒔𝒗𝒆𝒓𝒂𝒕𝒓𝒐𝒍)

𝒓𝒆𝒔𝒗𝒆𝒓𝒂𝒕𝒓𝒐𝒍 𝒍𝒐𝒂𝒅∗ 𝟏𝟎𝟎 (eq 3.1)

3.3.2.6 Resveratrol Recovery Measurements

Resveratrol was extracted from the microcapsules using methanol coupled with

sonication and volume standardization. A Qsonica probe sonicator (Cole Parmer, Vernon Hills,

IL) was used to sonicate 10 mg resveratrol microcapsules mixed with 4 mL methanol in a 15 mL

round bottom tube. The sonication probe was 3.22 mm and 50% amplitude was used. Sample

tubes were placed in ice water, during sonication for three cycles of 30 sec continuous

sonication followed by 30 sec no sonication, in order to reduce heat buildup of samples. The

solutions were transferred to volumetric flasks and brought up to 25 mL in order to account for

methanol evaporation during the sonication process. Samples were filtered with 0.2 µM filters

before HPLC analysis. Three measurements of each spray dried replication were taken for

resveratrol recovery. Resveratrol recovery was calculated using the following equation:

Resveratrol Recovery (%) = 𝒓𝒆𝒔𝒗𝒆𝒓𝒂𝒕𝒓𝒐𝒍 𝒆𝒙𝒕𝒓𝒂𝒄𝒕𝒆𝒅

𝒓𝒆𝒔𝒗𝒆𝒓𝒂𝒕𝒓𝒐𝒍 𝒍𝒐𝒂𝒅∗ 𝟏𝟎𝟎 (eq 3.2)

3.3.2.7 Fluorescence

Fluorescence was used to indirectly measure the binding between resveratrol and

sodium caseinate. The method was adapted from prior research measuring resveratrol and

sodium caseinate binding [35]. SpectraMax M2e Microplate Reader (Molecular Devices,

Sunnyvale, CA) was used to measure fluorescence with an excitation of 280 nm and emission

of 350 nm. Five replications of each sample were taken. An aliquot of 200 µl was loaded into

each well of a black, 96-well polystyrene plate (Costar, Corning Incorporated, Corning, NY).

3.3.2.8 Fluorescence of Resveratrol and Ethanol

Protein is able to fluoresce due to natural fluorophores in the protein, which are aromatic

amino acids [41]. When protein is bound to resveratrol, the protein no longer fluoresces. The

change in fluoresce has been used to calculate the amount of protein bound to resveratrol [35,

36]. Background fluorescence for all components was measured in order to control from non-

42

specific signals from components in the sample other than protein. Fluorescence measurements

ranged from 175 to 250 for various ethanol concentrations and from 110 to 250 for various

resveratrol concentrations solubilized in 2.5% EtOH (Figure 3.2). The average fluorescence of

a blank well was 243 (±7). Therefore, it was assumed that the fluorescence of ethanol and

resveratrol alone do not significantly contribute to the overall fluorescence of the samples which

fluorescence measurements ranged between 1200 and 2000. Therefore, the fluorescence of

resveratrol was not taken into account for the actual samples and it was assumed that the

decrease in fluorescence as resveratrol concentration increase was due to binding between the

protein and resveratrol.

3.3.2.9 Fluorescence of Solution with Spiked Resveratrol with Various Resveratrol Concentrations

Sodium caseinate solutions were prepared in the same manner as the resveratrol

microcapsules using partial denaturation of the protein. The resveratrol stock solution was 500

µg resveratrol/100 µl 50% EtOH (w/w). Resveratrol was spiked into 1 mg/mL sodium caseinate

solution at the following levels: 12.5, 25, 50, 100, and 200 µg/mL sodium caseinate solution

(0.055, 0.110, 0.219, 0.438, and 0.876 µM). The standard curve encompassed 8

concentrations of sodium caseinate: 0.007, 0.016, 0.031, 0.063, 0.125, 0.250, 0.500, and 1

mg/mL. The ratio of resveratrol:sodium caseinate was the same as the 4.8% (dry basis, w/w)

resveratrol microcapsule.

3.3.2.10 Fluorescence of Microcapsules with Varying Resveratrol Concentrations

When testing the fluorescence of microcapsules with varying resveratrol concentrations,

standard curves were built from sodium caseinate microcapsules without any resveratrol

content at 7 dilutions. The concentrations of sodium caseinate in the standard curve were

31.25, 62.5, 125, 250, 500, 1000, and 2000 µg/mL of sodium caseinate. Resveratrol

microcapsules were produced with various concentrations of resveratrol: 1.2%, 2.4%, 4.8%,

9.1%, 16.7% (% in final product, w/w). All sodium caseinate microcapsules in solution and

43

resveratrol microcapsule in solution were stored at 5°C overnight to allow protein to fully hydrate

before fluorescence measurements.

3.3.2.11 Stern-Volmer Equation

The Stern-Volmer equation was developed by Otto Stern and Max Volmer and it

indicates fluorescence quenching, which is a decrease in quantum yield of fluorophore

fluorescence due to interaction with a quencher molecule [42, 43]. This equation was modeled

after a simple mechanism utilizing one fluorophore, one quencher, one excited state and an

irreversible quenching mechanism [44]. A linear plot of the equation indicates a single class of

fluorophores while a curvature indicates complex formation of both static and dynamic binding

[43]. This equation has been used to measure binding affinity between flavonoids and bovine

serum albumin and resveratrol and proteins [35, 36, 43]. The Stern-Volmer equation is as

follows:

Stern-Volmer equation = 𝑭𝟎

(𝑭𝟎−𝑭) =

𝟏

𝒇 +

𝟏

𝒇

𝟏

𝒌𝒔𝒗

𝟏

[𝑸] (eq 3.3)

F0 represents the fluorescence of protein without resveratrol and F represents

fluorescence of protein with resveratrol. Ksv is the quenching constant and f is the fraction of

binding sites. [Q] is the concentration of resveratrol. The absolute concentration of protein and

resveratrol was calculated for each sample. The linear equation from the standard curve of

protein was used to determine the F0 of each sample by plugging the absolute concentration of

protein (x) and finding the fluorescence measurement (y). The f, fraction of binding sites, was

obtained from the best linear fit of 1/[Q] (M-1) on the x-axis and F0/(F0-F) on the y-axis (Figure

3.3). The concentration of sodium caseinate was multiplied by f to calculate the concentration

of resveratrol bound to protein.

3.3.2.12 Equation Based on Number of Resveratrol Binding Sites

The three amino acids that are able to fluoresce are tryptophan, tyrosine and

phenylalanine [35]. It is thought that tryptophan is mainly responsible for the fluorescence of

44

protein because tyrosine is easy quenched and phenylalanine has low quantum yield thereby

causing emission from these amino acids to be very low [35, 41]. Therefore, tyrosine and

phenylalanine have negligible effects on the fluorescence of proteins. It can be assumed that

the binding between resveratrol and tryptophan is responsible for the decrease in fluorescence

when resveratrol is present with protein.

The absolute amount of resveratrol and sodium caseinate was calculated for each

sample. The average molecular weight used for sodium caseinate was 22,814 g/M which was

derived from the molecular weight of each fraction and the percentage of each fraction in

sodium caseinate. The percentage by weight of each fraction of α(S1), α(S2), β-casein and κ-

casein in sodium caseinate used for the average molecular weight were respectively: 40%,

10%, 35%, and 15%. Amino acid sequences were found on the Uniprot database and

sequences from Bos taurus (bovine) were used [45]. The reference codes in the database for

α(S1), α(S2), β-casein and κ-casein were P02662, P02663, P02666 and P02668, respectively.

The molecular weight and number of tryptophan in each fraction were as follows: 23,000 g/M

and 2 tryptophan for α(S1)-casein, 25,230 g/M and 2 tryptophan for α(S2)-casein, 24,000 g/M

and 1 tryptophan for β-casein, and 19,000 g/M and 1 tryptophan for κ-casein. The molecular

weight, percentage of each fraction in sodium caseinate and the number of tryptophan in each

protein fraction were taken from the literature and it is important to note that these are

assumptions.

The number of tryptophan in each sodium caseinate fraction along with the percentage

of each fraction in the total protein were the factors that were taken into account when deriving

the resveratrol binding site equation (shown in Table 3.2). Therefore, the assumption of this

equation is that tryptophan is the only binding site of resveratrol. The molecular weight of each

protein fraction was also taken into account when deriving the equation. For each protein

fraction, the percentage of each protein fraction in the total protein was divided by the molecular

weight of the protein fraction and the resulting value was multiplied by the number of tryptophan

45

in the protein fraction. The sum of this value was calculated for each protein fraction and the

sum is the constant term in the resveratrol binding sites equation for sodium caseinate, shown

below.

Fraction of bound resveratrol = 𝟔.𝟓𝟏𝟖𝟖𝑬−𝟓∗[𝑷]

[𝑹] (eq 3.4)

[P] = concentration of sodium caseinate (g/L)

[R] = molar concentration of resveratrol (M)

Constant = # 𝑜𝑓 𝑡𝑟𝑦𝑝𝑡𝑜𝑝ℎ𝑎𝑛 𝑝𝑒𝑟 𝑝𝑟𝑜𝑡𝑒𝑖𝑛 𝑚𝑜𝑙𝑒𝑐𝑢𝑙𝑒

𝑚𝑜𝑙𝑒𝑐𝑢𝑙𝑎𝑟 𝑤𝑒𝑖𝑔ℎ𝑡 =

1.49

22,814 𝑔/𝑀 = 6.5188E-5 (M/g)

The resveratrol binding sites equation was also developed for whey protein. The

fractions of whey protein used in the equation were β-lactoglobulin, α-lactalbumin, and serum

albumin which were respectively: 66%, 25% and 9% of the total protein. Molecular weights and

number of tryptophan units in each fraction were 18,300 g/M and 2 tryptophan for β-

lactoglobulin, 14,200 g/M and 4 tryptophan for α-lactalbumin, and 66,400 g/M and 2 tryptophan

for serum albumin [46-48]. The molecular weight, percentage of each fraction in whey protein

and the number of tryptophan in each protein fraction were taken from the literature and it is

important to note that these are assumptions. The number of resveratrol binding sites for whey

protein was calculated using the following equation:

Fraction of bound resveratrol = 𝟏.𝟒𝟓𝟐𝟕𝑬−𝟒∗[𝑷]

[𝑹] (eq 3.5)

[P] = concentration of whey protein (g/L)


Constant = # 𝑜𝑓 𝑡𝑟𝑦𝑝𝑡𝑜𝑝ℎ𝑎𝑛 𝑝𝑒𝑟 𝑝𝑟𝑜𝑡𝑒𝑖𝑛 𝑚𝑜𝑙𝑒𝑐𝑢𝑙𝑒

𝑚𝑜𝑙𝑒𝑐𝑢𝑙𝑎𝑟 𝑤𝑒𝑖𝑔ℎ𝑡 =

2.64

18,173 𝑔/𝑀 = 1.4527E-4 (M/g)

3.3.2.13 UVA Stability Measurements

Solutions of 10 mg resveratrol microcapsules and 4 mL NanopureTM water filtered

through a Barnstead water purification system (Waltham, MA) were prepared in 15 mL tubes.

46

The solutions were exposed to UVA light with a wavelength of 365 nm using a Benchtop 2UV

Transilluminator (LM-20E, Ultra-Violet Products Ltd, Upland, CA). Samples were placed directly

on top of the UVA light source. This amount of UVA light translates to about 3.7 min of sunlight.

Resveratrol was extracted from the samples, in the same way, as the resveratrol recovery

measurements. Resveratrol isomers were quantified on HPLC and the ratios of trans:cis

resveratrol were calculated.

3.3.2.14 In-Vitro Digestion Test

Digestive stability and bioaccessibility of trans-resveratrol was evaluated using a 3-

phase digestion model simulating oral, gastric and intestinal phases; utilizing a modified method

from Green and others (2007) [49]. Twenty-five mg of microcapsules were used for each

measurement and 3 measurements were taken for each spray dried replication. The oral phase

consisted of the addition of 8 mg of α-amylase (Sigma A3176) to each sample and tubes were

placed in a 37ºC water bath at 90 rpm for 10 min. Base solution for the oral phase consisted of

potassium chloride (1.792 g/L), sodium phosphate (1.776 g/L), sodium sulfate (1.140 g/L),

sodium chloride (0.596 g/L) and sodium bicarbonate (3.388 g/L). For the gastric phase, 20 mg

of pepsin (Sigma P7125, final concentration 0.5 g/L in sample) in 2 mL of 0.1 M HCl was added

and samples were acidified to pH 2.5 using 1 M HCl (~200 µl). Sample volumes were brought

up to 40 mL with saline and tubes were placed in a shaking water bath for 1 hr at 37ºC. For the

intestinal phase, 2 mL 0.1 M NaHCO3 solution containing 40 mg pancreatin (Sigma P1750, final

concentration 0.8 g/L) and 20 mg lipase (Sigma L3126, final concentration 0.4 g/L) and 3 mL 0.1

M Na HCO3 solution containing 120 mg bile (Sigma B8631, final concentration 1.8 g/L) were

added to each sample. The pH of samples was adjusted to 6.5 using 1 M NaHCO3 (<50 µl, if

needed) and sample volumes brought up to 50 mL with saline (0.9% NaCl). Samples were

placed in the water bath for 2 hr at 37ºC. An aliquot of the resulting sample was taken and this

was referred to as the digesta. In order to isolate the aqueous fraction, the remaining sample

was centrifuged at 10,000 g for 1 hr (Allegra X-22R, Beckman Coulter, Brea, CA) and filtered

47

with 0.2 µM filters. Samples were placed immediately on ice between phases and blanketed

with nitrogen before incubations and storage to minimize oxygen in the sample tubes. Digestive

stability is the amount of resveratrol left in the sample after the completion of the small intestinal

phase (digesta) in comparison to the amount of trans-resveratrol loaded into the microcapsules

(resveratrol load).

Digestive stability (%) = 𝒕𝒓𝒂𝒏𝒔−𝒓𝒆𝒔𝒗𝒆𝒓𝒂𝒕𝒓𝒐𝒍 𝒊𝒏 𝒅𝒊𝒈𝒆𝒔𝒕𝒂

𝒓𝒆𝒔𝒗𝒆𝒓𝒂𝒕𝒓𝒐𝒍 𝒍𝒐𝒂𝒅 * 100% (eq 3.6)

Bioaccessibility is the experimentally determined estimate of what is available for intestinal

absorption by virtue of being solubilized in the continuous aqueous fraction of the digesta. This

was the amount of resveratrol partitioned into the micellar fraction, after high speed

centrifugation at 10,000 x g for 60 min. It was the comparison of the amount of trans-resveratrol

in the aqueous fraction in comparison to the resveratrol load.

Bioaccessibility (%) = 𝒕𝒓𝒂𝒏𝒔−𝒓𝒆𝒔𝒗𝒆𝒓𝒂𝒕𝒓𝒐𝒍 𝒊𝒏 𝒂𝒒𝒖𝒆𝒐𝒖𝒔 𝒇𝒓𝒂𝒄𝒕𝒊𝒐𝒏

𝒓𝒆𝒔𝒗𝒆𝒓𝒂𝒕𝒓𝒐𝒍 𝒍𝒐𝒂𝒅 * 100% (eq 3.7)

HPLC was utilized to measure the amount of trans-resveratrol in each digestive fraction.

Samples were filtered with 0.2 µM filters (VWR, Radnor, PA) before HPLC analysis.

3.3.2.15 Statistical Analysis

Formulations presented in Table 3.1 were spray dried in duplicates. Microsoft Excel

(Microsoft, Redmond, WA) was used to analyze data related to resveratrol-protein binding which

includes the equation of the best fit line of fluorescence data, calculating percentage of bound

resveratrol, and fraction of binding sites. Microencapsulation efficiency, resveratrol recovery,

UVA stability, digestion analysis, moisture content and water activity were completed in

triplicates. The software, Statistical Analysis System (Cary, NC), was used to conduct analysis

of variance (ANOVA, p<0.05) and least significant difference (LSD) testing on the data.

48

3.4. Results and Discussion

3.4.1 Moisture Content and Water Activity Measurements

Moisture content and water activity of the microcapsule formulations are represented in

Table 3.3. Moisture content was not significantly different between spray dried replications.

When whey protein was used to encapsulate avocado oil through spray drying, the moisture

content ranged from 2.24-2.89% with a standard deviation up to about 1% [50]. These results

were comparable to the moisture content found in our study when using whey protein

concentrate with the inclusion of fat (2.36-2.54%). Water activity of all resveratrol microcapsule

formulations varied between 0.06-0.15. This range of water activity was lower than that found

for bifidobacteria encapsulated in whey protein-based microcapsules in which the water activity

was between 0.14-0.18 [51]. The variation in water activity may be attributed to the different

encapsulation technique (spray drying vs. freeze drying) and the type of core material, as the

optimum condition for bifidobacteria is a water activity between 0.11-0.22 [52].

3.4.2 Morphology and Particle Size

Scanning electron microscope (SEM) images of resveratrol microcapsules showed that

the microcapsules had a particle size between 1-20 µm and they were non-spherical (Figure

3.4). They appear to have a wrinkled or dehydrated appearance which may be attributed to the

nature of the spray drying process when using protein as an encapsulation matrix. Void space

was created at the core of the spray dried particle due to expansion and shrinkage during the

spray drying process. Other research supports that spray drying with protein as a wall material

will produce this type of morphology [53, 54]. Morphology images of dried egg and skimmed

milk showed similar morphology as the resveratrol microcapsules [53]. Skimmed milk particles

were found to be hollow with an outer shell thickness between 20-60 µm. In addition,

morphology of pea protein microcapsules produced through spray drying had a similar

morphology to the resveratrol microcapsules [54].

49

3.4.3 Microencapsulation Efficiency

Microencapsulation efficiency measurements of the samples are shown in Table 3.4.

Whey-protein-concentrate-based microcapsules had a higher microencapsulation efficiency

than sodium-caseinate-based microcapsules (p<0.05). Furthermore, the inclusion of anhydrous

milk fat led to a decrease in microencapsulation efficiency (6%) in whey-protein-based

microcapsules only (p<0.05).

The microencapsulation efficiency of resveratrol was studied within liposomes and a

significant difference was found between the encapsulation methods tested [55]. Liposomes

prepared through sonication had 44-56% microencapsulation efficiency and those prepared

through extrusion had 92-96% microencapsulation efficiency. The low microencapsulation

efficiencies of liposomes produced through sonication may have been due to the high shear

force used in this processing method which probably resulted in resveratrol movement out of the

microcapsule. Microencapsulation efficiency of resveratrol within a solid lipid nanoparticle was

64-89% and within a nanostructured lipid carrier was 65-77% [56]. Our results were similar to

those found in the nanoparticles and nanostructured lipid carriers. Differences in

microencapsulation efficiencies between studies may be due to the level of solubility of

resveratrol in different mediums and the partitioning of resveratrol between these mediums.

3.4.4 Resveratrol Recovery

Recovery of resveratrol using sonication and methanol was not complete. Table 3.4

shows the recovery for most formulations was less than 66% after probe sonication. Therefore,

this suggests that the decreased recovery may be attributed to nonspecific binding between the

resveratrol and protein. Prior research supported that proteins can bind with resveratrol. The

binding constants of whey protein and sodium caseinate with resveratrol are 1.7 x 104 - 1.2 x

105 M-1 and 3.7 x 105 M-1 - 5.1 x 105 M-1, respectively [35, 36]. The binding between resveratrol

and whey protein was thought to be due to dipole-dipole and Van der Waal forces [36]. For

sodium caseinate, hydrogen bonding and hydrophobic interactions were thought to be

50

responsible for resveratrol-protein-binding [35]. The binding between resveratrol and sodium

caseinate was investigated, utilizing two equations: Stern-Volmer equation and resveratrol

binding sites equation. It was thought that the low resveratrol recovery from the microcapsules

was attributed to resveratrol-protein binding.

3.4.4.1 Fluorescence

Fluorescence of the proteins decreased as the resveratrol concentration in the sample

increased (Figure 3.5). The change in fluorescence indicated that the resveratrol was binding

with the protein and binding increased as resveratrol concentration increased.

3.4.4.2 Estimated Resveratrol Binding Using Stern-Volmer Equation

The increased stability of encapsulated resveratrol may decrease the recovery of

resveratrol from the microcapsules. It was thought that the binding between resveratrol and

protein inhibits the compound from being fully recovered from the microcapsules using only

probe sonication. Resveratrol may be fully extracted from the microcapsules using enzymes

and other physical treatments. Thereby, the quantification of binding between resveratrol and

protein is necessary in order to provide an explanation for the limited recovery of resveratrol

using only probe sonication.

The Stern-Volmer equation (eq 3.3) was used to calculate resveratrol-protein binding.

An f value of 0.35 and Ksv of 22,727 was obtained when fluorescence was measured of the

resveratrol microcapsules (Table 3.6). When sodium caseinate solutions were spiked with

resveratrol and Stern-Volmer equation used, it was calculated that the f and Ksv values were

1.04 and 9,640, respectively. An f value of 1.20 and Ksv of 29,600 were reported in the literature

for sodium caseinate solutions with spiked resveratrol [35]. The f value reported in the literature

was close to that of our sodium caseinate solutions with spiked resveratrol but different from

that of our resveratrol microcapsules with varying resveratrol concentrations.

The f value is a component in the calculation for bound resveratrol, therefore, the

amount of bound resveratrol in microcapsules was significantly less than in protein solutions.

51

An f value slightly greater than 1 may be due to a combination of dynamic and static quenching

[35]. Therefore, it was assumed that the lower f value in the resveratrol microcapsules indicated

that there was limited dynamic binding while the higher f value in the sodium caseinate solutions

indicated a higher dynamic binding. In the protein solutions, there was more fluidity of

components within the matrix therefore providing more opportunity for binding. In comparison,

there was less fluidity within the microcapsules thereby resulting in less binding between the

protein and resveratrol. The resveratrol microcapsules also had a lower R2 than the sodium

caseinate solutions which suggests that the additional processing in these samples may result

in a higher variation in the fluorescence measurements of the samples. These results suggest

that the Stern-Volmer equation was more suitable to compare samples with both dynamic and

static binding. This is reasonable as the equation was originally modeled for dynamic binding

[57, 58]. Also, it would not be appropriate to compare samples of different nature (microcapsule

vs. protein solution). Although the encapsulated resveratrol was mixed with water, it was

assumed that the resveratrol and protein generally remain within the microcapsule as

resveratrol has limited solubility in aqueous solution.

3.4.4.3 Effect of pH on Stern-Volmer Calculations

Fluorescence measurements of the resveratrol microcapsules were measured with and

without pH adjustment. The pH of samples without buffer or pH adjustment ranged from 6.4 to

7.3. Phosphate buffered saline, 1x pH 7.4 (Quality Biological Inc, Gaithersburg, MD) was used

to buffer protein standards and resveratrol microcapsule samples. The pH was adjusted to 7.4

± 0.05 with 1 M HCl and 1 M NaHCO3.

The fraction of binding sites and percent bound resveratrol were 0.35 and 6% with pH

adjustment and 0.44 and 8% without pH adjustment (Table 3.7). Fluorescence procedures

utilized by other research groups to measure protein and resveratrol binding adjusted pH of

samples to be pH 7 [36, 37], while other research groups did not control the pH [35]. This

52

suggests that pH may not be a significant factor in samples within the narrow pH range of 6.4 -

7.3.

3.4.4.4 Estimated Resveratrol Binding Using Resveratrol Binding Sites Equation

Results from using the derived resveratrol binding sites equation to calculate the amount

of maximum binding potentials of resveratrol are shown in Figure 3.6. According to the

resveratrol binding sites equation, all the resveratrol in the 1.2% resveratrol microcapsule for

both whey protein concentrate and sodium caseinate had the potential to bind to the protein.

The equation estimated that there was more bound resveratrol in whey-protein-concentrate-

based microcapsules because there are more tryptophan binding sites per mole in comparison

to sodium caseinate.

The structures of whey protein and sodium caseinate may also play a role in resveratrol-

protein binding as it affects the ability of binding sites to be accessed by resveratrol. Sodium

caseinate has a more open configuration without any tertiary structure while whey protein is a

globular protein [59-61]. Although sodium caseinate may have few tryptophan per protein

molecule, the protein structure may allow a higher percentage of these binding sites to be

accessible. In comparison, whey protein has more tryptophan per protein molecule but less of

these binding sites are accessible to bind with resveratrol.

3.4.4.5 Compare the Resveratrol Binding Using Stern-Volmer Equation and Resveratrol Binding Sites Equation

Amount of bound resveratrol (%) = [𝑃]∗ 𝑓

[𝑅] * 100% (eq 3.8)

[P] = molar concentration of protein (M)


f = fraction of binding sites available

The amount of bound resveratrol was compared using the microcapsules containing

4.8% resveratrol (dry basis, w/w). The calculation of maximum binding potential of resveratrol

53

by tryptophan in the microcapsule differs significantly when using the Stern-Volmer equation (eq

3.3) (6%) and the resveratrol binding sites equation (eq 3.4) (26%) (Table 3.8). The difference

in the results may be due to the fact that one result was experimentally determined while the

other solely accounts for the number of tryptophan in the protein. In addition, the resveratrol

binding sites equation is the maximum binding potential of resveratrol to tryptophan while the

Stern-Volmer equation is not an indication of the maximum binding potential. In addition, the

molecular weight of the protein fractions are assumptions as crude systems were used to

measure resveratrol-protein binding. The Stern-Volmer equation compares change in

fluorescence of the protein in the presence and absence of the resveratrol. The change in

fluorescence was assumed to be due to binding of the protein that prevents the protein from

fluorescing. The resveratrol binding sites equation solely accounts for the number of tryptophan

as it is the main amino acid responsible for binding with resveratrol [35].

The assumptions of the resveratrol binding site equation were that 1) one resveratrol

molecule binds with each binding site, 2) only tryptophan is the binding site for resveratrol and

3) all tryptophan in the protein are available for binding.

3.4.4.6 Total Resveratrol Accounted For

Table 3.9 shows the total resveratrol accounted for according to resveratrol binding sites

equation for both sodium caseinate and whey-protein-concentrate-based microcapsules. The

compilation of both experimentally determined resveratrol recovery data and the estimation of

bound resveratrol from the resveratrol binding sites equation indicate that sodium caseinate

microcapsules have approximately 90% resveratrol accounted for and whey protein concentrate

microcapsules have about 97.5% resveratrol accounted for. The resveratrol that was

unaccounted for may be lost in processing of the microcapsules broken down to metabolites.

3.4.4.7 Binding of Resveratrol and Protein

The stability of resveratrol was enhanced by the binding of resveratrol and protein. It

was found that specifically β-lactoglobulin, can form a 1:1 complex with resveratrol, binding to

54

the surface of the β-lactoglobulin with a binding constant of 104-106 M-1. The binding delayed

the conversion of trans to cis-resveratrol, thereby increasing the photo stability of resveratrol

[37]. Molecular docking research showed that the resveratrol was bound to the surface of β-

lactoglobulin by two hydrogen bond interactions [62]. One study compared different fractions of

whey protein and found that whey protein binds with resveratrol in a 1:1 complex, similar to the

results found of β-lactoglobulin [36]. The ratio of bound whey protein:resveratrol was

determined from fluorescence measurements. The binding constant of resveratrol and whey

protein was between 1.7 x 104 to 1.2 x 105 M-1 [36]. Binding between whey protein and

resveratrol was thought to be dipole-dipole and Van der Waal interactions. In sodium caseinate,

resveratrol was found to have a binding constant of 3.7 to 5.1 x 105 M-1. Binding between

sodium caseinate and resveratrol was thought to be hydrogen bonding and hydrophobic

interaction [35]. The binding constant of resveratrol with α-casein was 1.9 x 104 M-1 and with β-

casein it was 2.3 x 104 M-1 [63].

Our results differ from the literature as sodium caseinate was found in the literature to

have a higher binding constant than whey protein. In our results, we found that there was more

potential for resveratrol to bind with whey protein than sodium caseinate. The difference in

bound resveratrol was most likely because the resveratrol binding sites equation solely takes

into account the number of tryptophan within a protein. In addition, different testing conditions

such as temperature and ratio of resveratrol:protein were used among different researchers.

Our sample matrix was resveratrol microcapsules and due to the lack of dynamic binding in this

matrix, it may not be accurate to cross-compare our results to other research studies.

In addition, nonpolar interactions may be attributed to the binding between resveratrol

and protein. These interactions have been found between resveratrol and human islet amyloid

polypeptide oligomers and fibrils [64].

Overall, this research study showed a significant difference between the binding

calculations of the Stern-Volmer equation and the resveratrol binding sites equation. The Stern-

55

Volmer equation was experimentally determined through fluorescence while the resveratrol

binding sites equation takes into account the number of tryptophan in the protein. Whey protein

was shown to have a higher binding potential than sodium caseinate according to the

resveratrol binding sites equation, in order to increase resveratrol stability.

3.4.5 UVA Stability

The results from UVA stability tests are in the form of trans:cis resveratrol ratio as shown

in Table 3.4. Absolute amounts of trans- and cis-resveratrol in the samples were estimated

according to the ratios found after UVA light exposure (Table 3.5). Ratios of trans:cis

resveratrol were used to evaluate the stability because not all the resveratrol was able to be

recovered after the extraction. Therefore, the comparison of trans-resveratrol concentration

between formulations would not be a true representative of UVA stability. For this comparison,

it was assumed that the unrecovered resveratrol existed in the same trans:cis resveratrol ratio

as what was extracted. The results showed that sodium-caseinate-based microcapsules was

able to preserve the trans:cis resveratrol ratio in a higher proportion than the whey-protein-

concentrate-based microcapsules and unencapsulated resveratrol. This showed that sodium

caseinate is a better encapsulation material to maintain resveratrol bioactivity. UV stability of

resveratrol encapsulated with chitosan cross-linked with vanillin has been investigated [31]. The

recovery of trans-resveratrol after UV exposure of these microcapsules was about 78%. The

amount of recovered trans-resveratrol was maintained in the bioactive form and extracted from

the microcapsule. The UV light used in this study was 16 W at about 20 cm distance but the

condition used in our study was 8 W at 0 cm distance from the light source. The difference in

power and distance of the sample from the UV source may be attributed to differences seen in

the retention of trans-resveratrol between the studies but it is difficult to cross-compare the

studies because different UV light sources were used.

56

3.4.6 In-Vitro Digestions

Digestive stability of trans-resveratrol within the microcapsules (68-86%) was higher

than the unencapsulated resveratrol (47%) (p<0.05, Figure 3.7). The digestive stability was

generally higher in the sodium-caseinate-based microcapsules in comparison to the whey-

protein-concentrate-based microcapsules. In terms of bioaccessibility, all microcapsule

formulations increased bioaccessibility of resveratrol (48-60%) in comparison to unencapsulated

resveratrol (23%) (Figure 3.8). This suggests that the encapsulation of resveratrol decreased

the isomerization or degradation of resveratrol throughout digestion as evaluated by the in-vitro

digestion. Encapsulation may also have increased the solubility of resveratrol thereby playing a

factor in increased in-vitro stability. Sodium caseinate proved to be a better encapsulation

matrix than whey protein concentrate for resveratrol and the inclusion of anhydrous milk fat in

the formulation did not significantly affect stability. Therefore, it is not necessary to include

anhydrous milk fat in the formulation.

Encapsulation can aid in the protection of resveratrol throughout the digestion system

and enable targeted release of the compound in the human body. Our results suggested that

the bioavailability would be increased and degradation of resveratrol during digestion would be

decreased. This assumption is supported by the higher bioaccessibility and digestive stability of

the encapsulated resveratrol compared to unencapsulated resveratrol within the 3-phase in-vitro

digestion model.

When humans were fed 25 mg resveratrol orally, 70% of the compound was absorbed

but only trace amounts of trans-resveratrol were detected in the plasma. Most of the trans-

resveratrol was metabolized to sulfates and glucuronic acid found in the urine [21]. Research

by Azorin-Ortuno and others (2011) supported this theory as 50% of trans-resveratrol and

derived metabolites administered to pigs was found in the jejunum and ileum of the small

intestine [65]. Therefore, stabilization of resveratrol in the digestive tract is an important

57

consideration in order to deliver the compound in a bioactive form to the targeted part of the

digestive tract.

3.5 Conclusions

The resveratrol binding sites equation provides simple quantification of bound resveratrol

without experimentation. This method can be extended to other polyphenols or bioactive

compounds which bind to sodium caseinate and whey protein concentrate in a similar manner

as resveratrol. The equation can also be altered to fit the parameters of other types of protein

where the amino acid composition is known. The use of fluorescence and the resveratrol

binding sites equations have limitations as they only take into account binding of resveratrol with

aromatic amino acids. Further research can investigate if resveratrol can bind with additional

amino acids, aside from tryptophan or other components of the protein. This understanding will

provide a more accurate estimate of bound resveratrol and allow comparison of different

proteins as an encapsulation material.

The combination of spray drying as an encapsulation method and sodium caseinate as

an encapsulation matrix proved to be an effective approach to produce stabilized resveratrol.

This processing technique and encapsulation material are desirable as they are widely available

at relatively low cost. Thus, sodium-caseinate-based microcapsules can be further investigated

to increase stability in UV light and in-vitro digestion. One limitation of the findings was that the

microscopic images of the resveratrol microcapsules showed the morphology was non-

spherical. Future research can investigate the incorporation of specific components, such as

plasticizers, in resveratrol microcapsules and their effect on morphology and stability of

resveratrol within the microcapsule. The encapsulation-system-approach utilized in this

research can be extended to other labile, bioactive compounds such as quercetin or lutein to

increase their stability.

58

Partial denaturation of

protein

Two-step homogenization

Spray drying


Figure 3.1: Schematic diagram of resveratrol microcapsule production

Inlet temp. = 160ºC

Outlet temp. = 90ºC

Flow rate = 5-7 g/min

Air pressure = 5 kPa

Nozzle diameter = 0.7 mm

59

Figure 3.2: Fluorescence measurements from spectrofluorometer of: A) Ethanol concentrations without resveratrol, B) Various resveratrol concentrations solubilized in 2.5% ethanol

0

50

100

150

200

250

300

0% 2% 4% 6% 8% 10% 12%

Flu

ore

scen

ce M

easu

rem

en

t

Ethanol Concentration (%)

0

50

100

150

200

250

300

0 20 40 60 80 100

Flu

ore

scen

ce M

easu

rem

en

t

Resveratrol Concentration (µg/mL)

A)

B)

60

Figure 3.3: Best fit linear line of 1/[Q] (M-1) and F0/(F0-F), components of the Stern-Volmer equation. Fraction of binding sites calculated by 1/intercept of best fit line.

1/[Q] (M-1) and F0/(F0-F) from Stern-Volmer equation (eq 3.3) [Q]: Resveratrol concentration F0: Fluorescence measurement without resveratrol F: Fluorescence measurement in the presence of resveratrol

y = 1E-04x + 0.964R² = 0.9971

0

0.5

1

1.5

2

2.5

3

0 5000 10000 15000 20000

F0/(

F0-F

)

1/[Q] (M-1)

61

Figure 3.4: Scanning electron microscope images of resveratrol microcapsules

AMF: Anhydrous milk fat SCAMF: Sodium caseinate with AMF, SC: Sodium caseinate without AMF WPCAMF: Whey protein concentrate with AMF, WPC: Whey protein concentrate without AMF

SCAMF SC

WPCAMF WPC

62

Figure 3.5: Fluorescence measurements of sodium caseinate solutions with various resveratrol concentrations showing that as resveratrol increases, fluorescence decreases

y = 18773x-0.708

R² = 0.9903

0

500

1000

1500

2000

2500

3000

3500

0 50 100 150 200 250

Flu

ore

scen

ce M

easu

rem

en

t


63

Figure 3.6: Maximum binding potential of resveratrol in sodium-caseinate and whey-protein-concentrate-based microcapsules with various resveratrol concentrations according to resveratrol binding sites equation

WPC: Whey-protein-concentrate-based microcapsules SC: Sodium-caseinate-based microcapsules

100% 100%

58%

29%

15%

100%

52%

26%

13%7%

0%

10%

20%

30%

40%

50%

60%

70%

80%

90%

100%

1.2% 2.4% 4.8% 9.1% 16.7%

Maxim

um

Bin

din

g P

ote

nti

al

(%)

Resveratrol Concentration in Experimental Design (dry basis, w/w)

WPC

SC

64

Figure 3.7: Digestive stability (%) of resveratrol in microcapsules after 3-phase in-vitro digestion

Values represent average of two spray dried replicates Same letters represent not significantly different values (p<0.05, ANOVA and LSD) Anhydrous milk fat (AMF); sodium caseinate with AMF (SCAMF); sodium caseinate without AMF (SC); whey protein concentrate with AMF (WPCAMF); whey protein concentrate without AMF (WPC) * Unencapsulated resveratrol

86%

81%

71%68%

47%

0%

10%

20%

30%

40%

50%

60%

70%

80%

90%

100%

SCAMF SC WPCAMF WPC Control*

Dig

estive

Sta

bili

ty (

%)

a ab

bc c

d

65

Figure 3.8: Bioaccessibility (%) of resveratrol in microcapsules after 3-phase in-vitro digestion

Values represent average of two spray dried replicates Same letters represent not significantly different values (p<0.05, ANOVA and LSD) Anhydrous milk fat (AMF); sodium caseinate with AMF (SCAMF); sodium caseinate without AMF (SC); whey protein concentrate with AMF (WPCAMF); whey protein concentrate without AMF (WPC) * Unencapsulated resveratrol

60% 59% 58%

48%

23%

0%

10%

20%

30%

40%

50%

60%

70%

80%

90%

100%

SCAMF SC WPCAMF WPC Control*

Bio

acce

ssib

ility

(%

)

a a a b

c

66

Table 3.1: Formulas for resveratrol microcapsules using sodium caseinate and whey protein concentrate, with and without anhydrous milk fat

Component SCAMF SC WPCAMF WPC

Sodium Caseinate 80 g 80 g 0 g 0 g

Whey Protein Concentrate 0 g 0 g 80 g 80 g

Anhydrous milk fat 100 g 0 g 100 g 0 g

Deionized Water 920 g 920 g 920 g 920 g

Trans-Resveratrol (% by total solids)

9 g (4.8%) 4 g (4.8%) 9 g (4.8%) 4 g (4.8%)

67

Table 3.2: Percent of total protein, molecular weight, and number of tryptophan

for each protein fraction in A) sodium caseinate and B) whey protein concentrate

A)

Protein Fraction % of total protein by

weight Molecular Wt # of tryptophan

αS1-casein 40 23000 2

αS2-casein 10 25230 2

β-casein 35 24000 1

κ-casein 15 19000 1

Weighted Avg: 1.49

B)

Protein Fraction % of total protein by

weight Molecular Wt # of tryptophan

B-lactoglobulin 66 18300 2

alpha-lactalbumin 25 14200 4

serum albumin 9 66400 2

Weighted Avg: 2.64

68

Table 3.3: Percent moisture content and water activity, mean (±SD), of resveratrol microcapsules

Sample Moisture Content Water Activity

SCAMF R1 2.42a 0.12b

SCAMF R2 2.17ab 0.10d

SC R1 2.12ab 0.07g

SC R2 2.35a 0.11c

WPCAMF R1 2.36a 0.15a

WPCAMF R2 2.54a 0.06h

WPC R1 1.73bc 0.09e

WPC R2 1.51c 0.07f

(±standard deviation), R = replication AMF: Anhydrous milk fat SCAMF: Sodium caseinate with AMF, SC: Sodium caseinate without AMF WPCAMF: Whey protein concentrate with AMF, WPC: Whey protein concentrate without AMF Same letters within each column represent not significantly different values after ANOVA and LSD (p<0.05).

69

Table 3.4: Efficiency of microencapsulation, recovery and UVA stability ratio (Trans:Cis Ratio) of resveratrol in microcapsules

Sample Efficiency Recovery

UVA Stability

SCAMF 68%c 66%b 0.62a

SC 68%c 62%b 0.64a

WPCAMF 77%b 42%c 0.46b

WPC 83%a 37%c 0.39b

Control --- 93%a 0.49b

Values represent average of two spray dried replicates Same letters within each column represent not significantly different values after ANOVA and LSD (p<0.05). Anhydrous milk fat (AMF); sodium caseinate with AMF (SCAMF); sodium caseinate without AMF (SC); whey protein concentrate with AMF (WPCAMF); whey protein concentrate without AMF (WPC); Unencapsulated resveratrol (Control)

70

Table 3.5: Estimated absolute values of trans- and cis-resveratrol in the

resveratrol microcapsules after UVA light testing

Sample Trans:Cis resveratrol Ratio

Trans-resveratrol (µg)

Cis-Resveratrol (µg)

SCAMF 0.62 184 296

SC 0.64 187 293

WPCAMF 0.46 151 329

WPC 0.39 135 345

Control 0.49 158 322

Estimated absolute values are calculated according to the resveratrol load and ratio of trans:cis resveratrol after UVA light testing at 365 nm for 1 hour. Resveratrol load in 10 mg of microcapsules is 480 µg resveratrol. AMF: Anhydrous milk fat, Control: Unencapsulated resveratrol SCAMF: Sodium caseinate with AMF, SC: Sodium caseinate without AMF WPCAMF: Whey protein concentrate with AMF, WPC: Whey protein concentrate without AMF

71

Table 3.6: Comparison of the fraction of binding sites (f), quenching constant of sodium caseinate by resveratrol (Ksv) and correlation coefficient (R2) of resveratrol microcapsules, sodium caseinate solutions with spiked resveratrol, and values reported in the literature (similar procedure to sodium caseinate solutions with spiked resveratrol)

Sample f Ksv R2

Resveratrol microcapsules 0.35 22,727 0.897

Sodium caseinate solution with spiked resveratrol

1.04 9,640 0.997

Literature [32] at 25ºC 1.20 29,600 0.978

Values are components of the Stern-Volmer equation Table 3.7: Fraction of binding sites (f) and bound resveratrol in resveratrol microcapsules with and without pH adjustment to pH 7.4

Sample f Bound Resveratrol (%)

With pH adjustment (pH = 7.4) 0.35 6%

Without buffer or pH adjustment (~pH 6.4-7.3)

0.44 8%

f value is component of the Stern-Volmer equation

72

Table 3.8: Estimated maximum binding potential of resveratrol by tryptophan in microcapsule with various resveratrol concentrations using number of resveratrol binding sites in the protein

Sample Maximum Binding

Potential of Resveratrol by Tryptophan (w/w %)

Stern-Volmer Equation – Resveratrol Microcapsules

6%

Stern-Volmer Equation – Sodium Caseinate Solution with Spiked Resveratrol*

18%

Resveratrol Binding Site Equation 26%

* Ratio of resveratrol:sodium caseinate was the same as the 4.8% (dry basis, w/w) resveratrol microcapsule

Table 3.9: Total resveratrol accounted for according to resveratrol binding sites equation for 4 resveratrol microcapsule formulations

Sample Recovery1

Maximum Binding

Potential by Tryptophan2

Total Resveratrol

3

SCAMF 66% 26% 92%

SC 62% 26% 88%

WPCAMF 42% 58% 100%

WPC 37% 58% 95%

1Experimental resveratrol recovery using probe sonication 2Maximum binding potential of resveratrol by tryptophan according to resveratrol binding sites equation 3Total resveratrol (%) = Resveratrol recovery (%) + Maximum binding potential of resveratrol by tryptophan (%) SCAMF: sodium caseinate w/anhydrous milk fat microcapsule SC: sodium caseinate w/o anhydrous milk fat microcapsule WPCAMF: whey protein concentrate w/anhydrous milk fat microcapsule WPC: whey protein concentrate w/o anhydrous milk fat microcapsule

73

3.7 References





5. Opipari, A.W., et al., Resveratrol-induced autophagocytosis in ovarian cancer cells. Cancer research, 2004. 64(2): p. 696-703.



8. Pearson, K.J., et al., Resveratrol delays age-related deterioration and mimics transcriptional aspects of dietary restriction without extending life span. Cell metabolism, 2008. 8(2): p. 157-168.




12. Brown, V.A., et al., Repeat dose study of the cancer chemopreventive agent resveratrol in healthy volunteers: Safety, pharmacokinetics, and effect on the insulin-like growth factor axis. Cancer Research, 2010. 70(22): p. 9003-9011.



15. Hiroki, A. and J.A. LaVerne, Decomposition of hydrogen peroxide at water-ceramic oxide interfaces. The Journal of Physical Chemistry B, 2005. 109(8): p. 3364-3370.

16. Figueiras, T.S., M.T. Neves-Petersen, and S.B. Petersen, Activation energy of light induced isomerization of resveratrol. Journal of fluorescence, 2011. 21(5): p. 1897-1906.

17. Trela, B.C. and A.L. Waterhouse, Resveratrol: Isomeric molar absorptivities and stability. Journal of agricultural and food chemistry, 1996. 44(5): p. 1253-1257.



74

20. Iranshahy, M., et al., Method validation for the one-month stability study of trans–resveratrol in human plasma. Jundishapur Journal of Natural Pharmaceutical Products, 2013. 8(2): p. 65.


22. Meng, X., et al., Urinary and plasma levels of resveratrol and quercetin in humans, mice, and rats after ingestion of pure compounds and grape juice. Journal of agricultural and food chemistry, 2004. 52(4): p. 935-942.

23. Goldberg, D.M., J. Yan, and G.J. Soleas, Absorption of three wine-related polyphenols in three different matrices by healthy subjects. Clinical biochemistry, 2003. 36(1): p. 79-87.

24. F. Gibbs, S.K., Inteaz Alli, Catherine N. Mulligan, Bernard, Encapsulation in the food industry: A review. International journal of food sciences and nutrition, 1999. 50(3): p. 213-224.

25. Fang, Z. and B. Bhandari, Encapsulation of polyphenols–a review. Trends in Food Science & Technology, 2010. 21(10): p. 510-523.

26. Desai, K.G.H. and H. Jin Park, Recent developments in microencapsulation of food ingredients. Drying technology, 2005. 23(7): p. 1361-1394.

27. Munin, A. and F. Edwards-Lévy, Encapsulation of natural polyphenolic compounds; a review. Pharmaceutics, 2011. 3(4): p. 793-829.

28. Nam, J.B., et al., Stabilization of resveratrol immobilized in monodisperse cyano-functionalized porous polymeric microspheres. Polymer, 2005. 46(21): p. 8956-8963.

29. Lu, Z., et al., Complexation of resveratrol with cyclodextrins: Solubility and antioxidant activity. Food Chemistry, 2009. 113(1): p. 17-20.

30. Das, S., K.Y. Ng, and P.C. Ho, Formulation and optimization of zinc-pectinate beads for the controlled delivery of resveratrol. Aaps Pharmscitech, 2010. 11(2): p. 729-742.

31. Peng, H., et al., Vanillin cross-linked chitosan microspheres for controlled release of resveratrol. Food chemistry, 2010. 121(1): p. 23-28.

32. Pan, K., Q. Zhong, and S.J. Baek, Enhanced dispersibility and bioactivity of curcumin by encapsulation in casein nanocapsules. Journal of agricultural and food chemistry, 2013. 61(25): p. 6036-6043.

33. Pan, K., et al., Thymol nanoencapsulated by sodium caseinate: Physical and antilisterial properties. Journal of agricultural and food chemistry, 2014. 62(7): p. 1649-1657.

34. Sanguansri, L., et al., Encapsulation of mixtures of tuna oil, tributyrin and resveratrol in a spray dried powder formulation. Food & function, 2013. 4(12): p. 1794-1802.

35. Acharya, D.P., L. Sanguansri, and M.A. Augustin, Binding of resveratrol with sodium caseinate in aqueous solutions. Food chemistry, 2013. 141(2): p. 1050-1054.

36. Hemar, Y., et al., Investigation into the interaction between resveratrol and whey proteins using fluorescence spectroscopy. International Journal of Food Science & Technology, 2011. 46(10): p. 2137-2144.

37. Liang, L., H. Tajmir-Riahi, and M. Subirade, Interaction of β-lactoglobulin with resveratrol and its biological implications. Biomacromolecules, 2007. 9(1): p. 50-56.

38. Liang, L. and M. Subirade, Study of the acid and thermal stability of β-lactoglobulin–ligand complexes using fluorescence quenching. Food Chemistry, 2012. 132(4): p. 2023-2029.

39. Lopez, C., et al., Thermal and structural behavior of anhydrous milk fat. 2. Crystalline forms obtained by slow cooling. Journal of Dairy Science, 2001. 84(11): p. 2402-2412.

40. Picot, A. and C. Lacroix, Production of multiphase water‐insoluble microcapsules for cell microencapsulation using an emulsification/spray‐drying technology. Journal of Food science, 2003. 68(9): p. 2693-2700.

41. Lakowicz, J.R., Protein fluorescence, in Principles of fluorescence spectroscopy. 1983, Springer. p. 341-381.

75

42. Albani, J.R., Principles and applications of fluorescence spectroscopy. 2008: John Wiley & Sons.

43. Papadopoulou, A., R.J. Green, and R.A. Frazier, Interaction of flavonoids with bovine serum albumin: A fluorescence quenching study. Journal of agricultural and food chemistry, 2005. 53(1): p. 158-163.

44. Caldin, E., The mechanisms of fast reactions in solution. 2001: IOS Press. 45. Uniprot. 2014 [cited 2014 July 15]; Available from: http://www.uniprot.org/. 46. Foegeding, E.A. and S.W. Mleko, Whey protein products. 2002, Google Patents. 47. Edwards, P.B., L.K. Creamer, and G.B. Jameson, Structure and stability. Milk proteins:

From expression to food, 2009: p. 163. 48. Farrell Jr, H., et al., Nomenclature of the proteins of cows’ milk—sixth revision. Journal

of Dairy Science, 2004. 87(6): p. 1641-1674. 49. Green, R.J., et al., Common tea formulations modulate in vitro digestive recovery of

green tea catechins. Molecular nutrition & food research, 2007. 51(9): p. 1152-1162. 50. Bae, E. and S. Lee, Microencapsulation of avocado oil by spray drying using whey

protein and maltodextrin. Journal of microencapsulation, 2008. 25(8): p. 549-560. 51. Picot, A. and C. Lacroix, Encapsulation of bifidobacteria in whey protein-based

microcapsules and survival in simulated gastrointestinal conditions and in yoghurt. International Dairy Journal, 2004. 14(6): p. 505-515.

52. Celik, O. and D. O’Sullivan, Factors influencing the stability of freeze-dried stress-resilient and stress-sensitive strains of bifidobacteria. Journal of Dairy Science, 2013. 96(6): p. 3506-3516.

53. Walton, D. and C. Mumford, The morphology of spray-dried particles: The effect of process variables upon the morphology of spray-dried particles. Chemical Engineering Research and Design, 1999. 77(5): p. 442-460.

54. Costa, A., et al., Effective stabilization of cla by microencapsulation in pea protein. Food Chemistry, 2015. 168: p. 157-166.

55. Isailović, B.D., et al., Resveratrol loaded liposomes produced by different techniques. Innovative Food Science & Emerging Technologies, 2013. 19: p. 181-189.

56. Neves, A.R., et al., Novel resveratrol nanodelivery systems based on lipid nanoparticles to enhance its oral bioavailability. International journal of nanomedicine, 2013. 8: p. 177.

57. Parker, C.A. and C. Parker, Photoluminescence of solutions: With applications to photochemistry and analytical chemistry. 1968: Elsevier Amsterdam.

58. Bowen, E.J. and F. Wokes, Fluorescence of solutions. 1953. 59. Morr, C. and E. Ha, Whey protein concentrates and isolates: Processing and functional

properties. Critical Reviews in Food Science & Nutrition, 1993. 33(6): p. 431-476. 60. Siew, D.C., et al., Solution and film properties of sodium caseinate/glycerol and sodium

caseinate/polyethylene glycol edible coating systems. Journal of Agricultural and Food Chemistry, 1999. 47(8): p. 3432-3440.

61. Horne, D.S., Casein structure, self-assembly and gelation. Current opinion in colloid & interface science, 2002. 7(5): p. 456-461.

62. Sahihi, M., Z. Heidari-Koholi, and A.-K. Bordbar, The interaction of polyphenol flavonoids with β-lactoglobulin: Molecular docking and molecular dynamics simulation studies. Journal of Macromolecular Science, Part B, 2012. 51(12): p. 2311-2323.

63. Bourassa, P., J. Bariyanga, and H. Tajmir-Riahi, Binding sites of resveratrol, genistein, and curcumin with milk α-and β-caseins. The Journal of Physical Chemistry B, 2013. 117(5): p. 1287-1295.

64. Wang, Q., et al., Molecular mechanism of the inhibition and remodeling of human islet amyloid polypeptide (hiapp1-37) oligomer by resveratrol from molecular dynamics simulation. The Journal of Physical Chemistry B, 2014.

http://www.uniprot.org/

76

65. Azorín‐Ortuño, M., et al., Metabolites and tissue distribution of resveratrol in the pig. Molecular nutrition & food research, 2011. 55(8): p. 1154-1168.

77

CHAPTER 4: EFFECT OF PLASTICIZERS ON STABILITY OF ENCAPSULATED RESVERATROL

4.1 Abstract

Microencapsulation in a sodium-caseinate-based microcapsule has been shown to

provide higher UV stability of resveratrol compared to unencapsulated resveratrol, in our prior

research (Chapter 3). Scanning electron microscope images showed that the morphology of

the microcapsules was non-spherical, and this provided rationale to enhance the spherical

nature of the microcapsules through the addition of plasticizers. The increased spherical nature

would reduce surface area of the microcapsule thereby increasing stability of resveratrol.

Therefore, the objective of this research was to evaluate the effect of plasticizers (propylene

glycol, sorbitol, sucrose) on the UV stability of encapsulated resveratrol and morphology of the

microcapsules. It was found that the addition of plasticizers in the concentration range of 31.7-

47.6% (dry basis, w/w) to the resveratrol microcapsules decreased the trans:cis resveratrol ratio

after UV light exposure, thereby indicating a decreased light stability in comparison to the

formulation without added plasticizers. The UV light stability of encapsulated resveratrol within

a protein matrix with the inclusion of a plasticizer was not significantly different across the three

plasticizer types. Morphology images showed that the resveratrol microcapsules with sorbitol

used as a plasticizer exhibited a more spherical shape. The use of plasticizers also resulted in

the microcapsule wall being thin and fragile which may be related to the decreased light stability

of resveratrol in these microcapsules. Since the addition of plasticizers did not increase stability

of resveratrol nor enhance the spherical nature of the microcapsule, the secondary objective

was to evaluate the effect of protein denaturation on UV stability of resveratrol within a

microcapsule. The exclusion of protein denaturation in the preparation of the resveratrol

microcapsules resulted in a lower trans:cis resveratrol ratio after UV stability testing in

comparison to microcapsules in which the preparation included protein denaturation. The

stability of resveratrol within microcapsules without protein denaturation was not significantly

78

different than that of microcapsules with added plasticizers. Overall, the use of plasticizers and

absence of protein denaturation in the processing of resveratrol microcapsules did not enhance

the UV stability of the microcapsules.

Keywords: plasticizers, microencapsulation, resveratrol, stability, morphology

4.2 Introduction

Resveratrol (3,4’,5-trihydroxystilbene) is a polyphenol found in red grapes, red wine,

peanuts and blueberries [1-4]. This compound has been associated with health benefits related

to the prevention and alleviation of disease states related to cancer, diabetes, and heart disease

[5-8]. Some of the challenges with resveratrol incorporation into food systems are bitterness

and light instability [9-11]. Wines fortified with 20 and 200 mg resveratrol/L were found to have

an increased bitterness in comparison to wines without fortification [9]. In terms of light

instability, exposure of resveratrol to UV light for 1-2 hours isomerized 80-90% of resveratrol

from the bioactive form to the bio-inactive form [10, 11]. The incorporation of resveratrol into a

microcapsule with a stable wall material can help to overcome these challenges through the

innovative processing of resveratrol utilizing spray drying. Microencapsulation is readily used in

the food industry to protect micronutrients and also enable the delayed release of flavor

compounds [12, 13].

Our prior investigation was focused on the stability of resveratrol encapsulated in a

sodium caseinate matrix (Chapter 3). Encapsulation was able to increase both UV light and in-

vitro digestion stability of resveratrol. Scanning electron microscope images showed that the

resveratrol microcapsules were non-spherical and contained many folds and crevices. It was

hypothesized that the inclusion of plasticizers may further increase stability of the resveratrol by

enhancing the spherical shape of microcapsules thereby decreasing the surface area of the

compound that is exposed to environmental conditions.

79

The effect of plasticizers has been previously investigated in hydroxypropyl

methylcellulose (HPMC) based microcapsules produced through spray drying [14]. Plasticizers

that were used in the HPMC microcapsules were triethylcitrate, propylene glycol, polyethylene

glycol, glycerin and citric acid. Cohesiveness of the microcapsules was increased with the use

of plasticizers. Triethylcitrate microcapsule had a rapid release of the core material due to the

porous nature of the microcapsules. Propylene glycol, glycerin, and citric acid were shown to

have positive effects on microcapsule wall formation and drug release kinetics. Citric acid has

also been used as a plasticizer in theophylline particles with a cellulose polymer [15]. The

plasticizer addition increased the spherical nature of the particle and also the size of the drug

crystals.

There is a wide range of research that looked into the effect of plasticizers on film

formation that potentially can be translated to microcapsule formation. Sorbitol has been added

to sodium caseinate films in ratios of plasticizer to sodium caseinate ranging from 1:65 to 2:1

[16]. Partial specific volume of the sodium caseinate solution was reduced with the inclusion of

sorbitol, which indicated a more ordered structure of the protein. In addition, sorbitol led to a

decrease in glass transition temperature. The effect of plasticizers (sorbitol, glycerol,

polyethylene glycol) was investigated in fish protein films [17]. The use of sorbitol increased

mechanical resistance and decreased film flexibility. In comparison, glycerol and polyethylene

glycol exhibited a lower mechanical resistance and higher film flexibility. Sucrose had a more

significant effect as a plasticizer on elongation at break than invert sugar in cassava starch films

[18]. In β-lactoglobulin films, the effect of glycerol, sorbitol, polyethylene glycol, and sucrose

were compared according to the elastic modulus, tensile strength, and elongation percentage

[19]. The plasticizer efficiency in the films was ranked from most to least efficient as glycerol,

polyethylene glycol, sorbitol, and sucrose.

The objective of this study was to evaluate the effect of plasticizers on the stability of

resveratrol in sodium-caseinate-based microcapsules and the morphology of the microcapsules.

80

The three plasticizers that were compared in terms of UV stability and morphology were

propylene glycol, sorbitol, and sucrose. The hypothesis was that plasticizers will help to

enhance the spherical shape of the resveratrol microcapsules that would in turn increase the UV

stability of resveratrol within the microcapsule. The secondary objective was to evaluate the

effect of protein denaturation in the microcapsule processing on the UV stability of resveratrol in

microcapsules. The hypothesis was that protein denaturation would enhance UV stability of

resveratrol in the microcapsule by resulting in a more open protein structure thereby providing

more access to resveratrol binding sites.

4.3 Material and Methods

4.3.1 Materials

Propylene glycol (USP) had a purity of 99.8%, and sorbitol was a 70% solution

(USP/FCC); both were donated by Archer Daniels Midland (Chicago, IL, U.S.A.). Sucrose used

as a plasticizer was from C&H (Crockett, CA, U.S.A.). Sodium caseinate was donated by

Agropur (La Crosse, WI, U.S.A.), and resveratrol was donated by Dutch State Mines (DSM,

Parsippany, NJ, U.S.A.). All solvents used were HPLC grade and purchased from Sigma-

Aldrich (St. Louis, MO, U.S.A.). The internal standard used was oxy-resveratrol with >98%

purity from Cayman Chemicals (Ann Arbor, MI, U.S.A.). Filters, used before HPLC analysis,

were 0.2 uM PTFE (VWR, Radnor, PA, U.S.A.).

4.3.2 Microcapsule Production

The formulations of resveratrol microcapsules are shown in Table 4.1. All formulations

were spray dried in duplicates. Sodium caseinate solution was partially denatured for 2 hr in a

shaking water bath (C76 Water Bath Shaker, New Brunswick Scientific, Edison, NJ, U.S.A.) at

100 rpm and 80°C. Plasticizers were added to the microcapsules in a 1:1 ratio of protein to

plasticizer. This level of plasticizer was chosen because it was the average of prior studies that

used sorbitol and glycerol as plasticizers [16, 20]. Resveratrol and plasticizer were mixed into

81

the protein solution at 15,200 rpm with a hand mixer (IKA Works, Wilmington, NC, U.S.A.). The

resulting solution underwent 2 cycles of high-pressure homogenization (APV Gaulin Inc.,

Wilmington, MA, U.S.A.) at 55 MPa in order to create a homogeneous dispersion. A lab-bench

spray drier (B-290 Buchi Corporation, New Castle, DE, U.S.A.) was then used to produce

resveratrol microcapsules with an inlet temperature of 160°C and outlet temperature of 90°C.

Additional parameters of spray drying were flow rate between 5-7 g/min, air pressure of 5 kPa

and nozzle diameter of 0.7 mm. Aluminum foil was used to protect sample solutions from light

exposure during the microcapsule production process.

SC-SOR microcapsules with a 2:1 ratio of sodium caseinate to sorbitol was spray dried

with the same methodology as previously explained in this section. The purpose was to test if

the ratio of protein to plasticizer had a significant effect on resveratrol stability within the sodium-

caseinate-based microcapsule. In addition, SC microcapsules without protein denaturation

were also spray dried in order to observe the effect of protein denaturation on UV stability of

resveratrol within the microcapsule.


Scanning electron microscope (XL30 ESEM-FEG, FEI Company, Hillsboro, OR, U.S.A.)

at Beckmann Institute for Advanced Science and Technology (Urbana, IL, U.S.A.) was used to

observe the morphology of resveratrol microcapsules. In order to prevent surface charging,

gold-palladium through sputter coating (Desk-1 TSC, Denton Vacuum, Moorestown, NJ, U.S.A.)

was used to coat samples before viewing with a scanning electron microscope. Hivac mode

was used to observe the morphology of the resveratrol microcapsules at a voltage of 5 kV.

Particle size was estimated from the scanning electron microscope images.

4.3.4 Moisture Content and Water Activity

Moisture content and water activity were measured in three replications with HR 83

Halogen Moisture Analyzer (Mettler Toledo, Columbus, OH, U.S.A.) and Aqua Lab 4TE (Aqua

Lab Technologies, Riverside, CA, U.S.A.), respectively.

82

4.3.5 HPLC

Resveratrol isomers were quantified on high-performance liquid chromatography using

coulometric detection (ESA CoulArray detector, ThermoScientific, Sunnyvalle, CA, U.S.A.) and

a C18 column (Phenomenex Gemini 5u, 110A, 150x4.6 mm, Torrance, CA, U.S.A.). Standards

of both resveratrol isomers and oxy-resveratrol (Cayman Chemicals, Ann Arbor, MI) were used

to construct external standard curves. The HPLC method utilized a gradient of two mobile

phases, 100% methanol (Sigma-Aldrich, St. Louis, MO) and 25 mM sodium acetate (Sigma-

Aldrich, St. Louis, MO), pH 4.5. The starting condition was 30% methanol which was held for 2

min and gradually increased to 60% methanol at 14 min. These conditions were held for 3 min,

and the amount of methanol was decreased back to 30% within 2 min and held for an additional

2 min.

4.3.6 UV Stability

Ten mg of resveratrol microcapsules was added to 4 mL ultra-filtered water, to form a

solution, in 15 mL centrifuge tubes (Thermo-Scientific, Rochester, NY, U.S.A.). Samples were

exposed to 365 nm of UVA light for 1 hr in Benchtop 2UV Transilluminator (LM-20E, Ultra-Violet

Products Ltd., Upland, CA, U.S.A.). Resveratrol was extracted from the microcapsules with

probe sonication (Qsonica probe sonicator, Cole Palmer, Vernon Hills, IL, U.S.A.), using 3

cycles of 30 sec continuous sonication then 30 sec no sonication. The amplitude used in the

sonication was 50%, and the diameter of the probe was 3.22 mm. Sample tubes were placed in

ice water during probe sonication in order to minimize heat buildup within the samples.

Samples were filtered and then injected to HPLC to quantify resveratrol isomers. The ratio of

trans:cis resveratrol was compared among microcapsule formulations to evaluate UV stability of

the encapsulated resveratrol. UV stability testing was conducted in triplicates for each spray

dried replicate.

83

4.3.7 Statistics

Statistical analysis system (SAS, Cary, NC, U.S.A.) was used to conduct analysis of

variance (ANOVA) and least significant difference testing (LSD) on the data. Microsoft Excel

(Microsoft, Redmond, WA) was used to calculate the trans:cis resveratrol ratio of samples after

UV light exposure.

4.4 Results and Discussion


Scanning electron microscope images of resveratrol microcapsules are shown in Figure

4.1. Particle size for resveratrol microcapsules with sorbitol or sucrose used as a plasticizer

ranged between 1-30 µm. Resveratrol microcapsules with propylene glycol as a plasticizer

ranged between 1-20 µm. Sorbitol, as a plasticizer, helped to improve the spherical shape of

the microcapsules in comparison to the formulation without the addition of plasticizer. Sucrose

and propylene glycol had a minimal effect on the microcapsule morphology. Microcapsule walls

appeared to be thin and fragile, according to the microscopic images at 10,000-20,000

magnification (Figure 4.2). In films, the addition of plasticizers has been shown to increase

tensile strength that can decrease flexibility of the film thereby resulting in a brittle film [21].

Propylene glycol as a plasticizer in β-lactoglobulin films resulted in a brittle film and the

concentration of plasticizer was independent of mechanical strength [19]. Therefore, it is

hypothesized that the addition of plasticizers in the resveratrol microcapsules caused the

capsule wall to be weak and easily broken. The wall flexibility was probably decreased and

tensile strength increased, resulting in a capsule that was more rigid and broken with moderate

force. The walls may have been easily broken in the spray drying process as the particle

expands and contracts onto itself due to the heat used in the drying process.

84

4.4.2 Moisture Content and Water Activity

Resveratrol microcapsules with propylene glycol as a plasticizer had a significantly

higher moisture content than the sodium-caseinate-based resveratrol microcapsules without any

added plasticizers (Table 4.2). The resveratrol microcapsules with added propylene glycol also

had the highest water activity out of all microcapsule samples. Propylene glycol has a lower

molecular weight than the other two plasticizers used in resveratrol microcapsules (sorbitol and

sucrose). Therefore, there were a larger number of propylene glycol molecules in the

microcapsules than the other plasticizers, thereby providing more opportunity for the plasticizer

to bind with the protein. This decreased water binding with the protein may have resulted in a

higher moisture content and water activity of the microcapsule.

In addition, the use of sorbitol in the resveratrol microcapsules in a 2:1 ratio of sodium

caseinate to plasticizer resulted in significantly lower moisture content than the sodium-

caseinate-based resveratrol microcapsules. The addition of glycerol in chitosan films has been

shown to have an increased moisture content with increasing concentration of the plasticizer,

which disagrees with the findings of our study [22]. This occurrence indicated that the use of

plasticizers can alter the moisture content and water activity of the matrix. Water sorption in

films has been shown to be affected by a range of factors including size of the plasticizer,

interaction between water molecules, plasticizers and polymers [23]. Some plasticizers may

increase the water activity or moisture content while other plasticizers may decrease these

measurements.

4.4.3 UV Stability

The trans:cis resveratrol ratios of microcapsules with added plasticizers after UV light

exposure was significantly lower than microcapsules without plasticizers and unencapsulated

resveratrol (Figure 4.3). Therefore, the addition of plasticizers to resveratrol microcapsules

decreased the trans:cis resveratrol ratio after UV exposure. The effect of plasticizers in tara

gum films have been studied, and it was found that the plasticizers increased mobility of the

85

polymers within the film by decreasing intermolecular forces [24]. These results suggest that

the addition of plasticizers in the resveratrol microcapsules caused an increased mobility of

molecules that in turn may result in a higher concentration of resveratrol migrating to the surface

of the microcapsule.

It has been found that the size, shape and composition of a plasticizer can affect the

ability of a protein film from forming hydrogen bonding [19]. Therefore, incorporation of

plasticizers into the resveratrol microcapsules may have affected the stability of the compound

as the plasticizer binds with the protein, thereby reducing the number of binding sites available

to resveratrol.

There was no significant difference between the type of plasticizer used in the

microcapsule in terms of UV stability of resveratrol. Sorbitol and sucrose have been previously

compared as plasticizers in protein films, and it was found that sorbitol was a more effective

plasticizer in terms of elasticity, tensile strength and elongation [19]. In our research, the

plasticizers were compared only on the basis of UV stability that is aligned with evaluating the

light stability of resveratrol. The parameters measured in the protein films with added

plasticizers may not be directly correlated to UV stability of resveratrol in protein-based

microcapsules.

When the amount of sorbitol in the microcapsule was decreased to a 2:1 ratio of protein

to plasticizer, the trans:cis resveratrol ratio did not change significantly from microcapsules with

a 1:1 ratio of protein to plasticizer (Figure 4.3). The ratio of sodium caseinate to sorbitol did not

significantly affect the UV stability of resveratrol within the microcapsules. In addition, the lack

of denaturation of sodium caseinate significantly decreased the trans:cis resveratrol ratio after

UV exposure in comparison to the sodium-caseinate-based microcapsule with protein

denaturation. Therefore, protein denaturation plays a significant role in enhancing UV stability

of resveratrol within a protein microcapsule. Denaturation of protein has been defined as “a

major change from the original native structure, without alteration of the amino acid sequence”

86

[25]. Thus, the change in the native structure of sodium caseinate resulted in an increased UV

stability, perhaps by increasing the number of binding sites available on the protein for

resveratrol to bind.

4.5 Conclusions

Plasticizers have been shown to increase tensile strength of protein gels but when they

were applied to microcapsules with the objective of protecting resveratrol, they were not able to

increase stability of the compound. Tensile strength and other parameters commonly measured

in films may not be directly related to enhanced stability of bioactive compounds in microcapsule

matrixes. The use of stability testing is the most direct means to evaluate the effect of additional

components in microcapsules. Moisture content and water activity are thought to be factors that

affect the stability of these bioactive compounds, due to their effect on mobility of the compound

within the matrix. A limitation of this study was the effect of plasticizers was only investigated in

sodium-caseinate-based microcapsules. In order to protect resveratrol from environmental

factors, the incorporation of plasticizers into other types of protein-based microcapsules can be

further investigated in the future. Another limitation of this research was the morphology of the

microcapsules were only qualitatively evaluated by scanning electron microscope imaging and

future research can evaluate morphology through a quantitative measure. In addition, it is

important to note that the morphology of the particles may be different with scale up using a

larger spray dryer.

87


Figure 4.1: Scanning electron microscope images of sodium-caseinate-based resveratrol

microcapsules with and without plasticizers

SC: sodium caseinate without plasticizer SC-PG: sodium caseinate with propylene glycol SC-Sor: sodium caseinate with sorbitol SC-Suc: sodium caseinate with sucrose

SC

SC-SOR

SC-PG

SC-SUC

88

Figure 4.2: Scanning electron microscope at 10,000x and 20,000x magnification of

resveratrol microcapsules with added plasticizers

SC-SOR

SC-SOR

SC-SUC

SC-Sor: sodium caseinate with sorbitol SC-Suc: sodium caseinate with sucrose

89

Figure 4.3: Trans:Cis resveratrol ratio of resveratrol microcapsules with and without

plasticizers in comparison to unencapsulated resveratrol (Resv)

SC-PG: sodium caseinate with propylene glycol (1 protein:1 plasticizer) SC-Suc: sodium caseinate with sucrose (1 protein:1 plasticizer) SC-Sor: sodium caseinate with sorbitol (1 protein:1 plasticizer) SC-Sor 2:1: sodium caseinate with sorbitol (2 protein:1 plasticizer) SC Undenaturated: sodium caseinate without plasticizer, protein undenatured SC: sodium caseinate without plasticizer R: replication Resv: unencapsulated resveratrol

0.00

0.10

0.20

0.30

0.40

0.50

0.60

0.70

Trans:Cis

Re

sve

ratr

ol R

atio

a b

c c c cd cde def ef ef ef f

90

Table 4.1: Resveratrol microcapsule formulations with the addition of plasticizers and the

sodium-caseinate-based microcapsule without plasticizer (SC)

Component SC SC-PG SC-Sor SC-Suc

Sodium Caseinate 80 g 80 g 80 g 80 g

Plasticizer Purity --- 99.8% 70% ~100%

Plasticizer Amount --- 80 g 114 g 80 g

Deionized Water 920 g 920 g 886 g 920 g

Trans-Resveratrol (% by total solids)

4 g (4.8%) 8 g (4.8%) 8 g (4.8%) 8 g (4.8%)

SC: sodium caseinate without plasticizer SC-PG: sodium caseinate with propylene glycol SC-Sor: sodium caseinate with sorbitol SC-Suc: sodium caseinate with sucrose Sorbitol was a 70% solution with the remaining 30% being water. The purity of this plasticizer was accounted for in the microcapsule formulations.

91

Table 4.2: Resveratrol microcapsule analyses of mean moisture content (%) and mean

water activity of resveratrol microcapsules with the addition of plasticizers, sodium-

caseinate-based microcapsule without protein denaturation (SC-Undenat) and sodium-

caseinate-based microcapsule without plasticizer (SC)

Moisture Content Water Activity

SC-PG R1 3.14a 0.33a

SC-PG R2 2.87ab 0.27b

SC-Sor R1 1.67abc 0.08g

SC-Sor R2 2.81efg 0.18c

SC-Suc R1 1.88ef 0.10e

SC-Suc R2 1.70efg 0.06i

SC-Sor2:1 R1 1.61fg 0.08f

SC-Sor2:1 R2 1.34g 0.07g

SC-Undenat R1 2.53bcd 0.07h

SC-Undenat R2 2.41bcd 0.05j

SC R1 2.12de 0.07h

SC R2 2.35cd 0.11d

R = replication SC-PG: sodium caseinate with propylene glycol SC-Sor: sodium caseinate with sorbitol SC-Suc: sodium caseinate with sucrose SC-Sor 2:1: sodium caseinate with sorbitol (2 protein:1 plasticizer) SC-Undenat: sodium caseinate with plasticizer, protein not denatured SC: sodium caseinate without plasticizer

92

4.7 References












12. Han, J., et al., Alginate and chitosan functionalization for micronutrient encapsulation. Journal of agricultural and food chemistry, 2008. 56(7): p. 2528-2535.

13. Bhandari, B., et al., Flavor encapsulation by spray drying: Application to citral and linalyl acetate. Journal of Food Science, 1992. 57(1): p. 217-221.

14. Wan, L.S., P.W. Heng, and C.G. Chia, Plasticizers and their effects on microencapsulation process by spray-drying in an aqueous system. Journal of microencapsulation, 1992. 9(1): p. 53-62.

15. Wan, L., P. Heng, and C. Chia, Citric acid as a plasticizer for spray-dried microcapsules. Journal of Microencapsulation, 1993. 10(1): p. 11-23.

16. Barreto, P., et al., Effect of concentration, temperature and plasticizer content on rheological properties of sodium caseinate and sodium caseinate/sorbitol solutions and glass transition of their films. Food chemistry, 2003. 82(3): p. 425-431.

17. Bourtoom, T., et al., Effect of plasticizer type and concentration on the properties of edible film from water-soluble fish proteins in surimi wash-water. Food Science and Technology International, 2006. 12(2): p. 119-126.

18. Veiga-Santos, P., et al., Sucrose and inverted sugar as plasticizer. Effect on cassava starch–gelatin film mechanical properties, hydrophilicity and water activity. Food Chemistry, 2007. 103(2): p. 255-262.

19. Sothornvit, R. and J.M. Krochta, Plasticizer effect on mechanical properties of β-lactoglobulin films. Journal of Food Engineering, 2001. 50(3): p. 149-155.

93

20. Siew, D.C., et al., Solution and film properties of sodium caseinate/glycerol and sodium caseinate/polyethylene glycol edible coating systems. Journal of Agricultural and Food Chemistry, 1999. 47(8): p. 3432-3440.

21. Bourtoom, T., et al., Effect of plasticizer type and concentration on the properties of edible film from water-soluble fish proteins in surimi wash-water. Food Science and Technology International, 2006. 12(2): p. 119-126.

22. Fundo, J.F., et al., Molecular mobility, composition and structure analysis in glycerol plasticised chitosan films. Food chemistry, 2014. 144: p. 2-8.

23. Cheng, L.H., A.A. Karim, and C.C. Seow, Effects of water‐glycerol and water‐sorbitol interactions on the physical properties of konjac glucomannan films. Journal of Food Science, 2006. 71(2): p. E62-E67.

24. Antoniou, J., et al., Physicochemical and thermomechanical characterization of tara gum edible films: Effect of polyols as plasticizers. Carbohydrate Polymers, 2014.

25. Tanford, C., Protein denaturation. Advances in protein chemistry, 1968. 23: p. 121-282.

94

CHAPTER 5: TASTE DETECTION THRESHOLDS OF RESVERATROL

5.1 Abstract

Resveratrol is a polyphenol that is associated with numerous health benefits related to

heart disease, cancer, diabetes and neurological function. The addition of this compound to

food products would help to deliver these health benefits to the consumer. However, bitterness

associated with resveratrol may impart negative sensory qualities on the food products, which

may decrease consumer acceptability. This concern may be resolved by encapsulating

resveratrol through spray drying, an innovative processing technique. The objectives of this

research were to: 1) compare taste detection thresholds of unencapsulated resveratrol

(unencapsulated) and encapsulated resveratrol and 2) determine if the inclusion of anhydrous

milk fat in the formulation affects the taste detection threshold of resveratrol within the

microcapsules. Resveratrol microcapsules were produced by encapsulating resveratrol in a

protein matrix through spray drying. R-index measure by the rating method was used to find the

average taste detection threshold and the pooled group taste detection threshold. The average

and pooled group taste detection thresholds for resveratrol, sodium-caseinate-based resveratrol

microcapsule without fat (SC), and sodium-caseinate-based resveratrol microcapsule with fat

(SCAMF) were: 90 and 47 mg resveratrol/L, 313 and 103 mg resveratrol/L, 334 and 108 mg

resveratrol/L of resveratrol, respectively. The findings demonstrate that the encapsulation of

resveratrol decreased the detection of the compound and provided a means to incorporate the

resveratrol into food products without imparting negative sensory properties.

Keywords: Resveratrol, threshold, encapsulation, R-index, bitterness

95

5.2 Introduction

Resveratrol is one of the polyphenols found in the skin of red grapes and red wine,

which contributes to the health benefits of red wine [1, 2]. It has been shown to have positive

impacts on heart disease, cancer, diabetes and neurological function [3-6]. The addition of

resveratrol to food products would help to offer biologically active concentrations of the

compound in easy-to-consume foods in order to provide the health benefits to the consumer.

One of the challenges with the incorporation of resveratrol into products is the astringent

and bitter perception of the compound being a polyphenol [7]. However, there has been limited

research related to the sensory perception of resveratrol. Descriptive analysis testing was

utilized to evaluate bitterness of Riesling wine fortified with resveratrol [7]. A significant

difference in bitterness existed between wines fortified with resveratrol at 20 mg/L and the

control without any resveratrol. Significant differences in bitterness also existed between wines

fortified with 20 and 200 mg resveratrol/L, with the higher concentration being rated as more

bitter.

Chemical analysis on 23 Italian wines showed the concentration of resveratrol in wine

ranged from 0.18-5.44 mg resveratrol/L with the exception of one wine that had no resveratrol

content [8]. For the sensory evaluation of the Italian wines, a trained panel rated samples

according to a criteria commonly used for wine judging competitions according to the

Associazione Enotecnici Italiani-Organizzazione Nazionale Assagiatori Vini model which

provided one sensory evaluation score that took into account all categories such as

appearance, taste and bouquet. There was no direct correlation between the overall sensory

ratings and the resveratrol content. Overall sensory ratings were the only form of sensory

evaluation conducted and specific attributes were not evaluated individually.

There is very limited prior research on the sensory properties of resveratrol and none

that investigated the taste detection threshold of this compound. In addition, no research has

explored encapsulation of resveratrol as a way to mask the unattractive sensory perception of

96

resveratrol, which, in turn, could maintain consumer acceptance of products with added

resveratrol. In this research study, signal detection rating method was used to measure taste

threshold values of unencapsulated resveratrol and encapsulated resveratrol in solution. The

first objective of this research was to compare the taste detection threshold of resveratrol

microcapsules with a protein matrix to that of unencapsulated resveratrol. The hypothesis was

that the resveratrol within the microcapsules would have a higher taste detection threshold than

unencapsulated resveratrol. The second objective was to determine the effect of anhydrous

milk fat in the microcapsule formulation on taste detection threshold of resveratrol within the

microcapsule. Due to the higher fat solubility of resveratrol, the hypothesis was that the

resveratrol would be dispersed within the fat and the protein would form a protective layer

around the fat. Therefore, the taste detection threshold of resveratrol when encapsulated with

fat and protein combined was hypothesized to be higher than that of resveratrol microcapsules

that only utilized protein as a matrix. This research can provide an indication of the ideal level at

which encapsulated resveratrol microcapsules can be added to food products without a

negative impact on sensory properties of the product.


5.3.1 Encapsulated Resveratrol Production

Taste detection threshold was evaluated for two formulations of encapsulated

resveratrol. The first formulation contained 95.2% sodium caseinate (Agropur Ingredients, La

Crosse, WI, U.S.A.) and 4.8% resveratrol (DSM, Parsippany, NJ, U.S.A.) by solid weight basis.

The second formulation contained 52.9% anhydrous milk fat (Danish Maid, Chicago, IL, U.S.A.),

42.3% sodium caseinate and 4.8% resveratrol, by solid weight basis. The amount of protein in

the sample solution (8%) before spray drying was determined from prior research in our lab to

be a level at which the protein could be solubilized in solution [9]. The amount of anhydrous

milk was slightly higher than the amount of protein in the formulations. The increased amount of

97

fat was used to help solubilize the resveratrol within the solution [10]. Resveratrol concentration

was kept consistent among formulations at 4.8% resveratrol (dry basis, w/w). This resveratrol

concentration was chosen to provide more wall material than resveratrol.

Two formulations of resveratrol microcapsules were tested in order to compare the effect

of anhydrous milk fat in the microcapsule formulation on the taste detection threshold of

resveratrol. Sodium caseinate was partially denatured for 2 hours at 80ºC and 100 rpm in a

shaking water bath (C76 Water Bath Shaker, New Brunswick Scientific, Edison, NJ, U.S.A.).

Anhydrous milk fat and resveratrol were mixed into the protein solution using a hand mixer (IKA

Works, Wilmington, NC, U.S.A.) at 15,200 rpm for 3 min. An evenly dispersed solution was

obtained through a two-stage high-pressure homogenization (APV Gaulin Inc., Wilmington, MA,

U.S.A.) at 55 MPa. Resveratrol microcapsules were produced through spray drying with Buchi

B-290 (New Castle, DE, U.S.A.) spray dryer. The following spray drying conditions were used:

160ºC inlet temperature, 90ºC outlet temperature, 0.7 mm nozzle diameter, 4.5-7.5 g/min flow

rate and 7 kPa.

5.3.2 Sample Preparation

Unencapsulated resveratrol and the two types of encapsulated resveratrol were tested at

five concentrations, in three-fold increments. The concentrations of unencapsulated resveratrol

tested were 2.2, 6.7, 20, 60, and 180 mg resveratrol/L. The water solubility of resveratrol is

0.03 mg/mL; therefore, it is assumed that 30 mg/L of resveratrol is soluble and the remaining

resveratrol concentration is dispersed in solution [11]. The concentrations of resveratrol in the

microcapsules tested were 4.4, 13.3, 40, 120, and 360 mg resveratrol/L. These levels were

established by 2 rounds of preliminary testing with individuals who were familiar with threshold

testing. The first round of preliminary testing used 12 panelists (4 males and 8 females) and 1

replication of each concentration/panelist was tested. The concentrations tested for

unencapsulated resveratrol were 0.625, 1.25, 2.5 and 40 mg resveratrol/L. The concentrations

98

tested for encapsulated resveratrol were 0.625, 1.25, 2.5 and 80 mg resveratrol/L. The rinse

protocol consisted of 1) warm water and 2) room temperature water.

The second round of preliminary testing used 13 panelists (5 males and 8 females) and

two replications of each concentration/panelist were tested. The concentration tested for

unencapsulated resveratrol was 20 mg resveratrol/L and for encapsulated resveratrol was 40

mg resveratrol/L. The rinse protocol consisted of 1) room temperature water (Absopure,

Urbana, IL, U.S.A), 2) ice cream (Meijers, Grand Rapids, MI, U.S.A.), 3) carbonated water

(Meijers, Grand Rapids, MI, U.S.A.) and 4) room temperature water (Absopure, Urbana, IL,

U.S.A). The inclusion of the ice cream and carbonated water in the rinse protocol helped to

further cleanse the palette in comparison to the first testing.

In both preliminary tests, testing was conducted under incandescent lighting in a

conference room and responses were collected on paper ballots. The noise sample was 0.4%

ethanol solution for unencapsulated resveratrol and spring water for the resveratrol

microcapsules. The signal sample was the highest concentration of resveratrol being tested.

Panelists were first asked to familiarize themselves with both the signal and noise samples.

Once familiar with these samples, they were presented with the test samples and rated each

sample according to four options: 1) Signal Sure, 2) Signal Unsure, 3) Noise Unsure, and 4)

Noise Sure.

For the actual testing, six replications of the noise and each concentration were tested.

The noise sample was the background solution which unencapsulated resveratrol or the

microcapsules were dispersed within. In order to solubilize resveratrol, it was dissolved in 1.2%

ethanol (EtOH), 190 Proof, USP (New Brunswick, NJ, U.S.A.). It was confirmed by HPLC that

1.2% EtOH (v/v) was sufficient to dissolve 180 mg resveratrol/L (Appendix A). The

encapsulation of resveratrol helped to increase its solubility; therefore, it was not necessary to

use an ethanol solution for these samples. Resveratrol microcapsules without anhydrous milk

in the formulation were added to a base protein solution. The sodium caseinate added to the

99

base solution differed for each microcapsule concentration in order for the sum of protein in the

microcapsules and in the base solution to be equal to that in the highest concentration of

microcapsules. A similar procedure was applied to microcapsules with the inclusion of

anhydrous milk fat, the difference being that the base solution included both anhydrous milk fat

and protein, in order to keep the amount of protein and fat consistent across all microcapsule

concentrations. All base solutions underwent two-stage high-pressure homogenization (APV

Gaulin Inc., Wilmington, MA, U.S.A.) at 55 MPa in order to keep the solutions homogeneous.

All samples were prepared the day before testing, in a room without windows and under

amber light, in order to minimize light degradation of resveratrol. The wavelength associated

with yellow or amber light is the least degradative to light-sensitive resveratrol [12]. Twenty mL

of sample was placed in 60 mL clear, plastics containers with lids. Samples were put on trays,

and the entire tray was covered with foil and stored between 3-5°C, overnight. Samples were

labeled with a 3-digit code and served at room temperature.

5.3.3 Rating Method and R-index Measurement

R-index by rating method and the American Society of Testing and Materials (ASTM)

method of ascending limits are the most commonly used threshold methods [13-18]. R-index

measurement by rating has been used in relation to the noise and signal samples [19]. R-index

by rating method and ASTM method have been found to be similar when the thresholds of

caffeine solutions determined by the two methods were compared [13]. R-index by rating

method was proposed to be a more efficient method than ASTM, because fewer samples were

needed [13, 20].

5.3.4 Taste Detection Threshold Testing

This study utilized 33 panelists between 18-45 years of age, 11 males and 22 females.

Panelists were asked to not eat, drink, or smoke at least 30 minutes prior to the testing. They

were also instructed to not wear strong cosmetics that may interfere with their perception of the

100

samples. In the first session, panelists received a brief presentation on the testing method in

order to familiarize them with the rating method used to determine the R-index measure.

The testing was completed in a booth setting with red lighting, positive air flow, and room

temperature of 25ºC. Sample cups were placed on top of red colored paper on trays to help

mask color differences across the samples. Compusense® five Plus (version 5.6, Guelph ON,

Canada) was used to determine a randomized complete block design for six replications of five

concentration levels and the noise. Each panelist rated six replications of each concentration of

unencapsulated resveratrol and encapsulated resveratrol.

The taste detection threshold testing began with a warm-up phase, in which panelists

were presented with two samples: Noise and Signal. The noise sample was 1.2% ethanol for

unencapsulated resveratrol samples. For resveratrol microcapsules, the noise sample was the

base solution to which the microcapsules were added, as described in the sample preparation

section. The signal sample was the highest concentration of the sample being tested in the test

design. Panelists were instructed to swirl the samples on the tray for 2-3 seconds, then, place

the sample in their mouth and swish around for 2-3 seconds. After waiting 10 seconds, the

panelists were asked to focus on the perception of the sample, in order to fully assess

astringency which can be a delayed perception. Panelists rinsed their mouth with the same

rinse protocol before and between all samples. The rinse protocol was as follows: 1) heavy

whipping cream (Land O'Lakes, Arden Hills, MN, U.S.A.), 2) carbonated water (Meijers, Grand

Rapids, MI, U.S.A.), 3) room temperature water (Absopure, Urbana, IL, U.S.A). All samples and

rinses were expectorated.

Once the panelists were familiar with the Noise and Signal samples, they were

presented with the first sample tray which contained six samples (5 concentration levels of

resveratrol and one noise sample). The panelists rated all samples in comparison to the noise

and signal samples, choosing one of the four options: Signal Sure, Signal Unsure, Noise

Unsure, Noise Sure. The testing protocol of the samples was similar to the warm-up phase,

101

where panelists were instructed to swirl the cup on the tray before tasting and a 10-second timer

was embedded to the program to force panelists to wait 10-seconds before rating each sample.

Panelists were allowed to re-taste the noise and signal samples at any time.

After the completion of the first sample set, there was a 2-minute break, during which

time the panelists held ¼ of a slice of Sara Lee Soft and Smooth bread with no crust (Chicago,

IL, U.S.A.) in their mouth compressed between the top of the mouth and tongue to soak up any

residue sample or rinses. A built-in 2-minute timer in the Compusense® five Plus system

(version 5.6, Guelph ON, Canada) reinforced the break. After compressing the bread in the

mouth, panelists repeated the rinse protocol to cleanse the palate. Then, the panelists rated

another six samples that consisted of five concentration levels and the noise.

5.3.5 Testing of Additional Concentration Level

The empirical threshold is defined as 50% above the chance probability of selecting the

correct answer (R-index of 50%) [21], an R-index of 75% is the empirical threshold [13, 20, 22].

A number of panelists (11 panelists for unencapsulated resveratrol, 16 panelists for SC, 20

panelists for SCAMF) did not reach their individual threshold in the five concentration levels of

unencapsulated resveratrol and encapsulated resveratrol testing. The pooled R-index reached

above 75% only for the resveratrol testing range. Therefore, an additional testing was

completed, which included the highest concentration from the original testing, a concentration

that was 3-fold higher than the highest level from the original testing, and the noise to more

accurately determine the pooled and individual threshold values. Resveratrol was dissolved in

3.6% ethanol. Ethanol content was increased 3-fold from the original testing because the

concentration of resveratrol was tripled.

The additional testing was conducted in a similar manner to the original testing, but

panelists evaluated a total of 15 samples per session. Sample order was randomized using

Compusense® five Plus (version 5.6, Guelph ON, Canada) program, between and across all

sample sets. Five replications of each sample were tested with three sample trays of five

102

samples per tray. The same warm-up and rinse protocol were used in the additional testing.

Five samples were served on each tray with a 2-minute break between each sample tray, in

which panelists held ¼ of a slice of bread compressed between the tongue and top of the

mouth. After the 2-min break, panelists rinsed their mouth with the established protocol.

5.3.6 Post-Questionnaire

At the completion of the threshold testing, all panelists filled out a questionnaire that was

aimed at gathering 1) demographic information, 2) information on a suitable product for a health

claim related to resveratrol, and 3) knowledge of resveratrol. Some of the questions included:

"What product would be best aligned with the health claim, may decrease cancer risk, increase

heart health and neurological function?" and "Which of the following health benefits do you

associate with resveratrol?” Responses were collected on paper ballots, shown in Appendix B.

5.3.7 Data Statistical Analysis

Data were collected using Compusense® five Plus (version 5.6, Guelph ON, Canada).

This software assigned randomized sample orders to each panelist and presented the testing

questions along with the embedded timer to panelists throughout the testing. Data were

exported into Microsoft Excel (Microsoft, Redmond, WA, U.S.A.). Statistical Analysis System

(SAS, Cary, NC, U.S.A) was used to conduct analysis of variance (ANOVA) and least significant

difference (LSD) test on the data. Nonparametric LSD was used to find where the difference

existed between check-all-that-apply questions in the post questionnaire related to health

benefits of resveratrol.

5.3.8 Taste Detection Threshold Calculations

Taste detection thresholds were calculated for all pooled data and each panelist using

the R-index response matrix method [22]. For each panelist, resveratrol concentration (x-axis)

was plotted against the R-index percentage (y-axis). A linear line was constructed between the

two points above and below an R-index of 75%. Then, this linear equation was used to

calculate the resveratrol concentration (x-axis) at which the R-index (y-axis) was 75%. The R-

103

index of panelists who participated in the additional testing was calculated by the additional

testing data of the 180 mg resveratrol/L and 540 mg resveratrol/L in the microcapsules.

Panelists who did not have a taste detection threshold within the concentration range tested in

both the original and additional testing were not included in the average or pooled threshold

calculations. The sample size (N) in Table 5.1 indicates the number of panelists who had a

taste detection threshold within the tested concentration range. Pooled taste detection

thresholds (mg/L) were calculated by compiling the individual data from all panelists who

reached threshold in the first testing and data from the second testing of those who reached

threshold during the additional testing. The sample size for the pooled threshold was 198 (6

replications of each sample per panelist with a total of 33 panelists).


5.4.1 Taste Detection Threshold

The average individual panelist thresholds for encapsulated resveratrol were significantly

higher than unencapsulated resveratrol (Table 5.1). This indicated that the unencapsulated

resveratrol was detected at a lower concentration than resveratrol in the microcapsules. The

findings demonstrated that encapsulation of resveratrol within a protein or protein and fat matrix

successfully masked the detection of the compound in aqueous solutions. The presence of fat

within the formulation did not significantly decrease the detection of resveratrol as hypothesized,

which was evidenced by no difference between the thresholds of SC and SCAMF formulations

(P>0.05). These results were contrary to our expectations. The anhydrous milk fat is solid at

room temperature, which is the temperature that samples were served. Therefore, the

resveratrol would be physically encapsulated within the fat of the microcapsules. Opposed to

the microcapsules without anhydrous milk fat, in which resveratrol could be more easily

extracted from the microcapsules when in solution. One possible explanation is that the

resveratrol microcapsules were hollow as shown by scanning electron microscope images

104

(Chapter 3). Therefore, the water can diffuse into the microcapsules when mixed into a

solution. In this way, the resveratrol may diffuse into the aqueous phase and reach equilibrium

between the inside and outside of the microcapsule.

The pooled threshold of each sample was 2- to 3-fold lower than the average of the

individual panelists’ threshold (Table 5.1). In a past study on mouthfeel detection threshold for

sucrose and high fructose corn syrup (HFCS), the average of individual panelist thresholds and

pooled taste detection thresholds were similar to each other [14]. In comparison, the pooled

threshold of sulfur compounds in wines was 2- to 3-fold higher than the average individual

panelists’ threshold [23]. The discrepancy seen in our study between individual panelists’

average taste threshold and pooled threshold may have been due to the variation between the

propylthiouracil (PROP) status of panelists [24, 25]. PROP tasters may have been able to

detect the bitterness of resveratrol at a lower concentration than panelists who are not PROP

tasters. Therefore, the taste threshold of individuals who are PROP tasters would have a lower

individual taste detection threshold than panelists who are non-PROP tasters. The number of

replications to calculate the pooled taste threshold was 198, while the number of replications for

each sample for individual taste threshold was 6. The difference in threshold levels may be

attributed to the difference in the number of replications. The larger sample size used for the

pooled threshold may have minimized the variability across individual panelists [26]. Although

the pooled and average of the individual thresholds were different, the encapsulated resveratrol

had a higher threshold than the unencapsulated resveratrol in both the pooled and average

threshold results. Thereby, this supported that the effectiveness of the encapsulation of

resveratrol to mask the detection of the compound.

The number of panelists who had a taste detection threshold in the original testing for

unencapsulated resveratrol, SC, and SCAMF was 22, 17, and 13, respectively. These findings

suggested that as the complexity of the sample increased, the ability for the panelists to detect

the compound decreased. SCAMF had the most complex sample matrix with the inclusion of

105

resveratrol within sodium caseinate and anhydrous milk fat. It was assumed that the sodium

caseinate and milk fat contributed to the flavor notes of the samples that may have helped to

mask the bitterness or astringency of resveratrol. These results were in line with previous

research that compared the thresholds of pure limonin and naringin in different matrices which

included distilled water, sucrose solutions, citric acid solutions and citrus juice model systems

[27]. Limonin and naringin in a juice model system increased the threshold several folds in

comparison to the compounds in distilled water. Another study investigated the recognition

threshold of limonin and naringin in orange juice and found that the addition of sucrose to the

matrix decreased the bitterness of the compounds [28]. Therefore, an increased complexity of

the sample matrix can increase taste detection threshold of a compound within that matrix.

The increased threshold of resveratrol in the microcapsules also aligned with prior

findings which showed that the addition of sodium caseinate can lower the bitterness of olive oil

phenolics in an oil-free and 65% oil-in-water emulsion [29]. Our findings demonstrated that

encapsulation with sodium caseinate can mask bitterness of polyphenols in aqueous solutions.

Taste detection threshold testing using the R-index measure by rating method has been

completed for isoflavonoids such as genistein and daidzein [30]. The respective taste detection

thresholds for these compounds were 1080 mg/L and 740 mg/L. In addition, caffeine taste

detection thresholds have been found to be between 148-260 mg/L [13]. The taste detection

thresholds of resveratrol was lower than genistein, daidzein, and caffeine indicating the

importance of the use of innovative processing methods to decrease the detection of

resveratrol.

5.4.2 Post Questionnaire

Rank analysis in regards to the type of food product that would be best aligned with a

health statement related to resveratrol found that there was a significant difference across the

product choices. Yogurt had a significantly higher preference rank than all other products,

106

followed by drink and snack bars, which were not significantly different from one another.

Cookies had the lowest preference rank among all the food products.

When panelists were asked what health benefits they associated with resveratrol, “anti-

aging” was the most recognized (14 out of 33), followed by “prevent/treat cancer” (12 out of 33).

The option of “lower blood sugar levels” was selected the fewest number of times (5 out of 33).

“Anti-aging” was chosen significantly more than “lower blood sugar levels”, while the other

options were not significantly different from one another. The health benefits selected most

often were different than those normally associated with wine, which may indicate that the

panelists were not making a direct association between the health benefits of wine and

resveratrol. The findings suggested that panelists associated resveratrol as an anti-aging

compound. Thus, the ideal marketing of a product that contains resveratrol may need to

include the health benefits of the compound in order to show consumers the range of health

benefits associated with resveratrol.

5.5 Conclusions

This research sheds light on the taste detection threshold of resveratrol and

encapsulated resveratrol. The encapsulation of resveratrol significantly increased the taste

detection threshold of the compound in comparison to unencapsulated resveratrol, suggesting

that microencapsulation successfully masked taste or other sensory characteristics of the

compound. Therefore, the encapsulation of resveratrol provides the food industry a means to

add resveratrol into a wide range of foods products without negatively affecting sensory

attributes of the product. Use of spray drying as the processing method and sodium caseinate

as an encapsulation matrix are good options to produce resveratrol microcapsules due to the

wide availability and low cost of this method and ingredient.

One limitation of this study was that all samples were expectorated which is not a typical

way consumers would consume food products. The bitterness of resveratrol is often a delayed

107

perception which may be decreased when samples are not swallowed. We decided to have

panelists expectorate all samples in order to minimize fatigue and instead enforced a 10-second

timer before samples were rated. Yet, the results still demonstrated a significant difference

between taste detection of unencapsulated resveratrol and encapsulated resveratrol. Another

limitation was that this research only focused on encapsulation of resveratrol with sodium

caseinate as a wall material, and the findings from this study cannot be extrapolated to the taste

detection threshold of resveratrol encapsulated within other wall materials. Future research

could investigate the taste detection threshold of resveratrol encapsulated within different types

of encapsulation material such as cyclodextrins and gums. In addition, descriptive analysis

testing could be conducted in order to determine the specific sensory properties that may differ

between resveratrol and encapsulated resveratrol in solution and food products. This would

help to gain a better understanding of the effects of resveratrol on sensory properties when

added to food products.

108


Figure 5.1: Distribution of individual panelists’ taste detection thresholds of unencapsulated resveratrol, resveratrol encapsulated in SC (sodium caseinate) matrix, and resveratrol encapsulated in SCAMF (sodium caseinate with anhydrous milk fat) matrix

Total number of panelists who reached threshold was 27 for resveratrol, 26 for SC, and 22 for SCAMF. Resveratrol: unencapsulated resveratrol SC: sodium-caseinate-based microcapsule without anhydrous milk fat SCAMF: sodium-caseinate-based microcapsule with anhydrous milk fat

0

2

4

6

8

10

12

14

1.4-4.4 4.5-13 14-41 41-120 121-360 361-1080

Nu

mb

er

of

Pan

elists

Resveratrol Concentration (mg/L)

Resveratrol

SC

SCAMF

109

Figure 5.2: Check-all-that-apply results from health benefits associated with resveratrol

0 2 4 6 8 10 12 14

I do not know what resveratrol is

I am not aware of any of the health benefits…

Skin health

Prevent/treat cardiovascular disease

Prevent/treat high blood pressure/hypertension

Prevent/treat cancer

Lower blood sugar levels

Improved Neurological function

Anti-aging

Number of Responses

110

Table 5.1: Pooled threshold, average of individual panelist threshold, range of individual panelist thresholds and number of panelists who reached threshold in the concentration range tested (N), out of 33 total panelists in the taste detection threshold testing of resveratrol solutions

Resveratrol SC SCAMF

Pooled threshold 47 103 108

Average Individual Panelist Threshold

90A (±96) 313B (±286) 334B (±314)

Range of Individual Panelist Threshold

17-390 47-990 9.38-998

N 27 26 22

Threshold and range expressed in mg/L. Same letter superscripts for the average threshold represent not significantly different values (p<0.05). Resveratrol: unencapsulated resveratrol SC: sodium-caseinate-based microcapsule without anhydrous milk fat SCAMF: sodium-caseinate-based microcapsule with anhydrous milk fat

111

5.7 References








8. Lante, A., et al., Chemical parameters, biologically active polyphenols and sensory characteristics of some italian organic wines. Journal of Wine Research, 2004. 15(3): p. 203-209.

9. Kuo, W.Y. and Y. Lee, Temporal sodium release related to gel microstructural properties—implications for sodium reduction. Journal of Food Science, 2014. 79(11): p. E2245-E2252.

10. Hung, C.F., et al., Development and evaluation of emulsion-liposome blends for resveratrol delivery. Journal of Nanoscience and Nanotechnology, 6, 2006. 9(10): p. 2950-2958.

11. Aldrich, S., Resveratrol sigma prod. No. R5010. 1997. 12. Luo, Y.-W. and W.-H. Xie, Effect of germination conditions on phytic acid

andpolyphenols of faba bean sprouts (vicia faba l.). Legume Research, 2013. 36(6): p. 489-495.

13. Robinson, K., B. Klein, and S.-Y. Lee, Utilizing the r-index measure for threshold testing in model caffeine solutions. Food Quality and Preference, 2005. 16(4): p. 283-289.

14. Kappes, S., S. Schmidt, and S.Y. Lee, Mouthfeel detection threshold and instrumental viscosity of sucrose and high fructose corn syrup solutions. Journal of Food science, 2006. 71(9): p. S597-S602.

15. Argaiz, A., O. Pérez‐Vega, and A. López‐Malo, Sensory detection of cooked flavor

development during pasteurization of a guava beverage using r‐index. Journal of Food Science, 2005. 70(2): p. S149-S152.

16. Pipatsattayanuwong, S., et al., Hedonic r‐index measurement of temperature preferences for drinking black coffee. Journal of Sensory Studies, 2001. 16(5): p. 517-536.

17. Cliff, M., et al., Evaluation of r-indices for preference testing of apple juices. Food quality and preference, 1997. 8(3): p. 241-246.

18. CLIFF, M.A., et al., Development of a ‘bipolar’r‐index1. Journal of Sensory Studies, 2000. 15(2): p. 219-229.

19. Green, D.M. and J.A. Swets, Signal detection theory and psychophysics. Vol. 1974. 1966: Wiley New York.

112

20. Robinson, K., B. Klein, and S. Lee, Utilizing the r‐index measure for threshold testing in model soy isoflavone solutions. Journal of Food Science, 2004. 69(1): p. SNQ1-SNQ4.

21. ASTM.E.1432.2002, Standard practice for defining and calculating individual and group sensory thresholds for forced-choice data sets of intermediate size. Method e 1432, in Annual book of astm standards, S.R.a. others, Editor. 2002, ASTM: West Conshohocken, Penn. p. 75-82.

22. O'Mahony, M., Understanding discrimination tests: A user‐friendly treatment of response bias, rating and ranking r‐index tests and their relationship to signal detection. Journal of Sensory Studies, 1992. 7(1): p. 1-47.

23. Cliff, M., et al., Comparison of new and existing threshold methods for evaluating sulfur compounds in different base wines. Journal of Sensory Studies, 2011. 26(3): p. 184-196.

24. Lipchock, S.V., et al., Human bitter perception correlates with bitter receptor messenger rna expression in taste cells. The American Journal of Clinical Nutrition, 2013. 98(4): p. 1136-1143.

25. Drewnowski, A. and C.L. Rock, The influence of genetic taste markers on food acceptance. The American Journal of Clinical Nutrition, 1995. 62(3): p. 506-511.

26. BI, J. and D.M. ENNIS, Sensory thresholds: Concepts and methods. Journal of Sensory Studies, 1998. 13(2): p. 133-148.

27. Guadagni, D.G., V.P. Maier, and J.G. Turnbaugh, Effect of some citrus juice constituents on taste thresholds for limonin and naringin bitterness. Journal of the Science of Food and Agriculture, 1973. 24(10): p. 1277-1288.

28. Dea, S., et al., Interactions and thresholds of limonin and nomilin in bitterness perception in orange juice and other matrices. Journal of Sensory Studies, 2013. 28(4): p. 311-323.

29. Pripp, A.H., J. Busch, and R. Vreeker, Effect of viscosity, sodium caseinate and oil on bitterness perception of olive oil phenolics. Food Quality and Preference, 2004. 15(4): p. 375-382.

30. Robinson, K., B. Klein, and S. Lee, Utilizing the r‐index measure for threshold testing in model soy isoflavone solutions. Journal of Food science, 2008. 69(1): p. SNQ1-SNQ4.

113

CHAPTER 6: CONSUMER ACCEPTANCE OF BARS AND GUMMIES WITH RESVERATROL AND ENCAPSULATED RESVERATROL

6.1 Abstract

The addition of resveratrol, a polyphenol found in red wine and peanuts, to food products

would help to provide the health benefits associated with the compound to the consumer in a

wide array of food matrices. The bitterness of resveratrol and instability in light are two major

challenges with the incorporation of the compound into food products. In this research,

microencapsulation in a sodium caseinate matrix was utilized as a strategy to overcome these

challenges. The objective of this research was to show the application of the resveratrol

microcapsules in easy-to-consume foods. Consumer acceptance was evaluated for gummies

and bars with encapsulated resveratrol in comparison to the controls. Two concentrations of

resveratrol that have been shown to have therapeutic effects in humans were tested (10 and 40

mg/serving). The overall liking of bars with 10 mg of encapsulated resveratrol did not differ

significantly from the sample without any added resveratrol or protein or from bar samples with

equivalent protein and/or resveratrol concentrations. For gummies, the samples with the

resveratrol microcapsules had a significantly lower overall liking than samples with the same

protein and/or resveratrol content. The differing results across bars and gummies suggest that

a more complex matrix may help to mask the negative sensory properties of resveratrol. A

cluster of panelists was identified who preferred the bar samples with 10 mg encapsulated

resveratrol (SC 10) more than bar samples with the same amount of protein and

unencapsulated resveratrol (P+R 10) and the bar sample with the same protein concentration

only (PRO 10). The findings suggest that complexity of the food matrix may affect the masking

of negative sensory properties of resveratrol. Future research can identify which component of

the matrix (ex. fat, sugar) has the most significant effect on masking these properties.

Keywords: consumer testing, overall liking, CATA, resveratrol, microencapsulation

114

6.2 Introduction

Resveratrol is a polyphenol found in the skin of red grapes, red wine and peanuts [1-3].

This compound may be of interest to the food industry because it has been extensively shown

through both in-vitro and in-vivo studies to provide health benefits [4-10]. It has been

associated with reduction in blood glucose and improvement in plasma glucose in diabetic rats,

that indicates positive improvements related to diabetes [6]. Resveratrol has also been

demonstrated to prevent and slow down tumor growth, related to positive effects on cancer [11-

13]. In humans, it has been shown to inhibit platelet aggregation and increase cerebral blood

flow which decreases the risk of cardiovascular disease [8, 9, 14].

Due to the numerous health benefits associated with resveratrol, it would be valuable for

the food industry to incorporate the compound into easy-to-consume foods in order to deliver

the health benefits to consumers. Resveratrol could be incorporated into food products at levels

which have been shown to be biologically active. These levels are much higher than the levels

naturally found in foods with resveratrol [1-3]. One drawback is the instability of resveratrol in

the presence of light, in which about 90% of the bioactive form of resveratrol was converted to

the bio-inactive form of resveratrol after 100 min of light exposure at 366 nm [15]. Another

limitation with the incorporation of resveratrol into food products is the bitterness associated with

the compound. Riesling wine fortified with resveratrol at 20 and 200 mg/L were found to be

significantly more bitter than the control wine without added resveratrol [16]. Research on the

sensory properties of resveratrol is very limited and further research should be conducted to

gain a better understanding, especially related to consumer acceptance of food products with

added resveratrol.

Consumer testing can measure the acceptance of products with added resveratrol. This

type of testing uses untrained panelists who are familiar with the type of product being tested.

The product acceptance can be affected by attributes such as appearance, aroma, taste, and

texture, but it has been found that taste has the greatest influence on acceptance [17]. The 9-

115

point hedonic scale is most commonly used in consumer testing to measure degree of liking.

This type of scale is easy for panelists to use and easy for researchers to implement [18]. In

addition to overall liking questions using the 9-point hedonic scale, check-all-that-apply (CATA)

questions can be used in consumer testing to evaluate product attributes. There is some

disagreement on whether the use of CATA questions may bias the hedonic scales when used in

the same testing procedure. It has been found that CATA questions have less of an effect on

hedonic scale ratings in comparison to just-about-right and intensity scale ratings [19]. On the

other hand, it was found that the completion of attribute analysis along with hedonic ratings

biased overall ratings [20]. Research supported that rotating presentation order of CATA terms

or using a forced choice CATA format did not have a significant influence on hedonic scale

ratings [21]. Consumer testing using the 9-point hedonic scale along with CATA questions with

a rotating presentation order can be used to evaluate food products with added resveratrol.

Ratings from this testing can evaluate acceptance of products with encapsulated resveratrol in

comparison to the unencapsulated resveratrol.

The results of our prior research supported that resveratrol encapsulated within a

sodium caseinate matrix had a significantly higher stability (Chapter 3) and higher taste

detection threshold in comparison to unencapsulated resveratrol (Chapter 5). This indicated

that the encapsulation within a sodium caseinate matrix provided stability and masked negative

sensory attributes of resveratrol. Therefore, the objective of this research was to show

application of the resveratrol microcapsules to easy-to-consume food products and evaluate

consumer acceptance of these products. The hypothesis was that products with encapsulated

resveratrol will have a consumer acceptance not significantly different than the sample without

any added resveratrol or protein. The second hypothesis was that there will be a higher

consumer acceptance for products with encapsulated resveratrol in comparison to the product

with unencapsulated resveratrol.

116


6.3.1 Materials

Resveratrol and the sodium caseinate used were from Dutch State Mines (DSM,

Parsippany, NJ) and Agropur (La Crosse, WI), respectively. The oatmeal, almond milk and

distilled water were all Meijer® brand (Grand Rapids, MI). Nestlé® chocolate chips (Vevey,

Canton of Vaud, Switzerland) and Skippy® peanut butter (Austin, MN) were used for the bar

samples. Knox® gelatin (Kraft Foods, Chicago, IL), grape flavored Kool-Aid® (Kraft Foods,

Chicago, IL), and Jello-O® (Kraft Foods, Chicago, IL) were used for the gummy samples.

6.3.2 Encapsulated Resveratrol Production

Our previous research used 4.8% resveratrol (dry basis, w/w) in the microcapsules but

the concentration of resveratrol was increased for the consumer testing to minimize the amount

of microcapsules which needed to be added to the food products. The microcapsules used in

the consumer testing contained 9.1% resveratrol and 90.9% sodium caseinate (dry basis, w/w).

An 8% sodium caseinate solution was mixed, then partially denatured at 80°C for 2 hours in a

shaking water bath (C76 Water Bath Shaker, New Brunswick Scientific, Edison, NJ). The

resveratrol was mixed into the sodium caseinate solution before high-pressure homogenization

(APV Gaulin Inc., Wilmington, MA) at 55 psi, for two passes. The resulting solution was spray

dried in a lab bench spray drier (Buchi B-290, New Castle, DE). The inlet and outlet

temperatures for spray drying were 160°C and 90°C, respectively. The nozzle diameter was 0.7

mm and flow rate was 4.5-7.5 g/min, in order to maintain a constant outlet temperature.

6.3.3 Preparing Samples

Bar and gummy samples were prepared according to the formulations in Table 6.1. The

serving size for bars and gummies were 31 g and 25 g, respectively. These serving sizes were

averages of bars and gummies currently on the market.

117

6.3.4 Bars

For the bars, all ingredients except oatmeal were microwaved for 2 min on high, then

mixed by hand for 15 sec. The heating and mixing was repeated a second time before an

additional 1 min of heating. The resveratrol microcapsules, protein, and resveratrol were mixed

into the heated mixture until thoroughly dispersed. Then, the oatmeal was added. Fifteen

grams of the sample was weighed out and hand-shaped into circular bars. The samples were

put into the refrigerator for 30 min in order to set. Then, the bars were transferred to plastic

containers and placed in the refrigerator until the day before testing when samples were

transferred to 60 mL plastic cups with lids.

6.3.5 Gummies

All ingredients were combined and mixed together with a spatula. Then, the mixture was

microwaved for 2 min and 45 sec. The mixture was vortexed to evenly disperse the resveratrol

microcapsules, protein, and resveratrol in the mixture. The resulting solution was placed in a

45°C water bath for 30 minutes under vacuum in order to minimize air incorporated in the

solution. Gummies were molded in 1 cm x 1 cm x 1 cm cubes and placed in the freezer to

harden for 30 min. Samples were stored in the refrigerator for about 5-7 days in plastic

containers until the day before testing when samples were transferred to 30 mL plastic cups

with lids.

6.3.6 Resveratrol Dosage

For both bars and gummies, 10 and 40 mg resveratrol/serving were tested as these are

effective levels that align with the therapeutic dosages. Prior research has found 10 mg and 40

mg of resveratrol to be biologically effective in humans [10, 22, 23]. Resveratrol has been

shown to be effective in reducing insulin resistance and urinary ortho-tyrosine excretion related

to positive effects on diabetes at 10 mg/day and having an anti-oxidant and anti-inflammatory

effect related to anti-aging at 40 mg/day.

118

6.3.7 Panelists

There were a total of 100 panelists ranging from 18 to over 65 years of age. There were

35 males and 63 females reported. Two panelists did not answer the question regarding

gender. Panelists’ demographics are shown in Figure 6.1.

6.3.8 Consumer Testing

The testing took place in the Bevier Hall Spice Box, a large room where meals are

commonly served in the food science building. There were two days of testing in which

gummies were tested on the first day and bars were tested on the second day. Four different

controls were used for both gummies and bars: 1) without any resveratrol or protein (Plain), 2)

unencapsulated resveratrol (Resv), 3) sodium caseinate and unencapsulated resveratrol just

mixed without encapsulation (P+R), and 4) sodium caseinate only (PRO). The unencapsulated

resveratrol and sodium caseinate were added in the same concentration as the resveratrol

microcapsules which had a resveratrol concentration of 10 and 40 mg. Therefore, there were a

total of 9 samples for each of the testing days. Amounts of protein, resveratrol and

microcapsules added to each sample are shown in Table 6.2.

All samples were served to the panelists at once, in a multiple presentation format.

Samples were served at room temperature and covered with foil until being served to panelists.

Sample order was randomized among panelists using 25 different sample orders determined by

the RAND function in Microsoft Excel (Microsoft, Redmond, WA). All responses were collected

on paper ballots and a copy of the ballot can be found in Appendix C. Panelists were asked to

rate overall liking of each of the samples along with check-all-that-apply (CATA) questions

regarding what attributes they LIKE and DISLIKE about the samples. The attributes included in

the CATA questions were developed through preliminary testing with 7 panelists who were

experienced in generating descriptive attributes, specifically for these products. The order that

the CATA options were presented were randomized using 25 different orders.

119


Panelists were asked two questions related to health benefits and label claims related to

resveratrol: “Which of the following health benefits do you associate with resveratrol? (Check all

that apply)” and “If a product was labeled with “May decrease cancer risk, increase heart health

and neurological function”, would this increase the chance that you purchase this product over

another product without a health claim?” These questions were intended to gauge the panelists’

knowledge of resveratrol and purchase intent. Appendix D shows a copy of the post-

questionnaire.

6.3.10 Data Analyses

All responses were entered from the paper ballots into Microsoft Excel (Microsoft,

Redmond, WA). Statistical Analysis Software (SAS, version 9.3, SAS Inst. Inc., Cary, NC) was

used to analyze overall liking and CATA data. Analysis of variance (ANOVA) found if there was

a significant difference across average overall liking of samples and Fisher’s least significant

difference (LSD) test identified where the difference existed across average overall liking scores

of samples. The data generated from the overall liking scores was assumed to be continuous

data thereby allowing the use of ANOVA and LSD to analyze the data.

Cochran’s Q Equation, shown below, was used to calculate the significance level of

each attribute for the CATA data, separately for LIKE and DISLIKE, of each product.

Cochran’s Equation: Q= 𝒏𝒌 (𝒏𝒌−𝟏) ∑ (𝑻𝒌−𝑻)^𝟐𝒏𝑲

𝒌=𝟏

𝒏𝒌 ∑ 𝑹𝒋𝒏𝒋𝒋=𝟏 −∑ 𝑹𝒋^𝟐

𝒏𝒋𝒋=𝟏

(eq 6.1)

nk: number of products Tk: number of times attribute was chosen for specific product T: total number of times attribute was chosen across all products nj: number of panelists Rj: number of times attribute was chosen across all products by a specific panelist

Cochran’s Q-value was converted into the p-value using the Chi-squared distribution

function in Microsoft Excel. The nonparametric LSD was utilized to find where the difference

120

existed across samples in terms of the number of times an attribute was chosen for LIKE and

DISLIKE, separately. XLSAT 2014.4.09 (Addinsoft, New York, NY) was used for agglomerative

cluster analysis and correspondence analysis. Agglomerative cluster analysis grouped

panelists according to their overall liking scores of products with added resveratrol

microcapsules for gummies and bars, separately. Correspondence analysis provided a visual

depiction of the relationship of attributes to each other according to determined groups of

factors.


6.4.1 Overall Liking

The average overall liking scores for all samples are shown in Figure 6.2. There was no

significant difference in overall liking for bars when comparing samples with equivalent protein

and/or resveratrol content to SC 10. In addition, there was no significant difference in overall

liking between SC 10 bar and the Plain bar. These results indicated that the addition of 10 mg

of encapsulated resveratrol to bar samples did not significantly change the overall liking of the

product. For the gummies, samples with both concentrations of microcapsules had a

significantly lower overall liking in comparison to other samples with the equivalent

concentration of protein and/or resveratrol. This suggested that the complexity of the food

matrix affected overall liking scores across samples with encapsulated resveratrol and

unencapsulated resveratrol. Bars are complex food matrices and there was no significant

difference across SC and other samples, while there was a significant difference across SC and

other samples in gummies.

Panelists were clustered according to their ratings of samples with resveratrol

microcapsules (Figure 6.3 and Table 6.3). Therefore, there was similarity in overall liking

ratings of panelists within each cluster. The overall liking scores of the cluster of panelists who

rated SC samples highest in comparison to the other clusters of panelists (N=23 for bars) show

121

that, for bars, SC 10 had a significantly higher overall liking score than P+R 10 and PRO 10

(Figure 6.4 and Table 6.4). The panelists in this cluster liked SC 10 significantly more than

samples with the same protein content. For gummies, the overall liking scores of the cluster of

panelists who rated SC samples highest in comparison to the other clusters of panelists (N=28

for gummies) were not significantly different than other samples with the same resveratrol

and/or protein concentrations, for both concentrations of resveratrol microcapsule. Therefore,

the resveratrol microcapsules in gummies had no significant effect on overall liking in

comparison to other samples with the same protein and/or resveratrol concentration. Also, the

overall liking scores from this cluster of panelists for SC 10 and SC 40 bar and gummy samples

were not significantly different from the Plain samples. Thereby, this finding indicated that the

addition of encapsulated resveratrol maintained the overall liking of the sample.

6.4.2 Check-all-that-apply

The attributes that showed a significant difference (α=0.05) across the number of times

the attribute was chosen across products are shown in bold in Table 6.5. For bars, bitter and

sweet were significant attributes for both LIKE and DISLIKE. Sweetness has been shown to be

most resistant to being masked by other tastes [24]. Therefore, the significant differences seen

in both LIKE and DISLIKE are in line with the literature showing that sweetness is not masked

by changes in bitterness or sourness. Chalky and mouth coating were significant attributes for

both LIKE and DISLIKE in the gummies. This demonstrates that attributes related to taste

differentiated bar samples and attributes related to texture differentiated gummy samples.

Therefore, an increased complexity of the sample matrix may help to mask textural impacts of

added resveratrol microcapsules.

The results of the nonparametric LSD performed on the CATA data are shown in Table

6.6. The number of times bitter was chosen as a DISLIKE attribute for bars with added

resveratrol microcapsules was significantly higher than all other products with equivalent protein

and/or resveratrol concentrations. When comparing the non-parametric LSD for gummies, Resv

122

40 was selected significantly more times for chewy, mouth coating and smooth as a LIKE

attribute than SC 40. There was no significant difference across the number of times SC 40

was chosen as a LIKE attribute in comparison to PRO 40 and P+R 40 for chewy, mouth coating

and smooth. Therefore, the protein content in the sample was most likely attributed to the

difference in chewy, mouth coating and smooth for LIKE attributes of gummies. Sweet was

selected as a DISLIKE attribute the most for Plain, which was significantly higher than all

products with unencapsulated resveratrol and encapsulated resveratrol. These results are not

in agreement with prior research as it has been found that bitterness and sweetness were

negatively correlated so increased levels of bitterness resulted in decreased ratings of

sweetness [25]. Therefore, it would be expected that samples with resveratrol would have

sweet chosen as a DISLIKE option significantly more than samples without resveratrol. This

may be an indication that the Plain sample was the least sweet and consumers did not like it

because the sweetness was not on par with the other samples. The CATA questions indicate

differences between the number of times a specific attribute was chosen for LIKE and DISLIKE.

These questions do not indicate intensity of the attributes but descriptive analysis could be used

to accomplish this.

Complexity of the samples may be attributed to differences seen across the CATA

results of bars and gummies since the matrix of bars was more complex than that of gummies.

Discrimination testing between caffeinated and uncaffeinated solutions showed that ability to

discriminate between samples was affected by complexity of the beverage such as flavor,

sweetness and carbonation [26]. It has also been found that increasing the complexity of odor-

taste mixtures decreased that ability of trained panelists to identify specific components [27, 28].

According to correspondence analysis for the LIKE attributes of bars, the attributes that

were closely related to each other but separated from the other attributes were 1) bitter and

sour, 2) chalky and hard, and 3) astringent (Figure 6.5). For the LIKE attributes of gummies, the

attributes that were separated from the others but also not associated with each other were 1)

123

hard, astringent, foamy, bitter and 2) chalky. The attributes that were separated from the

internal cluster of attributes are the drivers of liking and disliking between the products. Future

research could utilize descriptive analysis to evaluate the intensity of specific attributes of the

bar and gummy samples, in order to confirm the findings from the correspondence analysis.

The overall findings of this study suggested that the complexity of the food matrix can affect

whether a difference exists across the overall liking of products with added resveratrol

microcapsules and the controls. Also, there may be a segment of consumers who prefer

products with encapsulated resveratrol in comparison to products with the same concentration

of protein and/or unencapsulated resveratrol. For this segment of consumers, it was also found

that overall liking of both bars and gummies with encapsulated resveratrol was maintained in

comparison to the Plain sample. The CATA data indicated that taste differentiated the bar

samples while texture distinguished gummy samples.


The post-questionnaire asked the panelists about prior knowledge of the health benefits

related to resveratrol and the effect of a health claim related to resveratrol on purchase intent.

For the questions related to prior knowledge of resveratrol, the option “I don’t know what

resveratrol is” was chosen that most, followed by “Prevent/treat cardiovascular disease” and

“anti-aging” (Figure 6.6). Also, most panelists indicated that the health claim related to

resveratrol would increase their purchase intent of the product. The results of the health claim

related to resveratrol showed that the claim would increase the chance that consumers would

purchase the product (Figure 6.7). Therefore, in order to positively influence the purchase intent

of consumers, it would be better to include health claims related to resveratrol rather than just

stating the resveratrol content since most panelists did not know of resveratrol.

124

6.5 Conclusion

This research provides a basis upon which the food industry can add stabilized

resveratrol into food products in order to deliver the health benefits to the consumer. The

benefit of using resveratrol microcapsules is that the encapsulation matrix helps to protect the

compound and maintain its bioactivity. The overall liking of SC 10 in bars was not significantly

different than that of the Plain sample. In addition, a group of panelist was identified who had a

higher overall liking for the SC 10 bar sample in comparison to the bar sample with both

unencapsulated resveratrol and protein (P+R 10) and the bar sample with equivalent amount of

protein (PRO 10). Therefore, these types of products with added resveratrol should be targeted

towards a segment of the market who prefer these products. Some limitations of this study

were that only two levels of resveratrol concentration and only two types of food products were

tested, as consumer acceptance of additional food matrixes may differ from those tested in this

research. Future research could evaluate consumer acceptance of other complex food matrices

utilizing additional resveratrol concentrations that have been shown to have therapeutic effects.

Another limitation of the study is that for the CATA questions, it is not possible to determine if

the panelists checked an attribute because it was present or not present. Therefore, descriptive

analysis would be helpful to determine attribute intensities of products with added resveratrol.

125


Figure 6.1: Demographics of panelists who participated in consumer testing of food products with added resveratrol: A) Ethnicity, B) Age (years of age)

There were a total of 100 panelists who participated in the consumer testing.

2%

45%

2%

41%

9%

1%

American Indian

Asian

Black

Caucasian

Hispanic

Native Hawaiian

44

38

11

3 2

1

18-25

26-35

36-45

46-55

56-65

>65

A)

B)

126

Figure 6.2: Average overall liking scores of panelists from the consumer testing of A) Bars and B) Gummies, with added unencapsulated resveratrol and encapsulated resveratrol.

Same letters represent not significantly different values (p<0.05, ANOVA and LSD) Average overall liking scores indicated above each sample bar Plain: no added resveratrol or protein, PRO 10: same sodium caseinate concentration as SC 10, PRO 40: same sodium caseinate concentration as SC 40, Resv 10: 10 mg of unencapsulated resveratrol, Resv 40: 40 mg of unencapsulated resveratrol, P+R 10: same sodium caseinate and resveratrol concentration as SC 10, P+R 40: same sodium caseinate and resveratrol concentration as SC 40, SC 10: 110 mg resveratrol microcapsule, SC 40: 439.6 mg resveratrol microcapsules.

5.66 5.625.16

5.63 5.68 5.43 5.344.94

4.31

1

2

3

4

5

6

7

8

9

Plain PRO 10 PRO 40 Resv 10 Resv 40 P+R 10 P+R 40 SC 10 SC 40

a a a ab a

a ab

b c

B)

5.82 5.896.19 6.07

5.285.81

5.385.73

4.99

1

2

3

4

5

6

7

8

9


A)

ab a a ab cd a bcd abc d

127

There were a total of 100 panelists who participated in the consumer testing. Figure 6.3: Cluster analysis of panelists based on overall liking ratings of samples with encapsulated resveratrol for: A) Bars and B) Gummies.

The concentration of encapsulated resveratrol within the food products was 10 and 40 mg/serving. The cluster with a box around it represents panelists who rated the samples with encapsulated resveratrol higher than the other two clusters of panelists. There were a total of 100 panelists who participated in the consumer testing. Panelists were clustered using agglomerative cluster analysis.

372 27 73 92 87 25 61 98 50 88 84 77 76 45 51 62 33 11 13 94 59 26 58 65 24

171 10

095 17 68 91 16 42 41 99 54 79 74 82 60 43 44 36 66 63 34

5 849 21

4 648 78 39 18 64 53

914 86 47 30 31 40 56 19 46 90 85 81 12 55 93 89 52 23 10 22 83 69

720 97 32 80 15 38 96 70 67 29 35

0

10

20

30

40

50

60

70

80

90

Dis

sim

ilari

ty

B)

128

Figure 6.4: Average overall liking scores for cluster of panelists who rated samples with resveratrol microcapsules highest for: A) Bars (N=23) and B) Gummies (N=28)

Same letters represent not significantly different values (p<0.05, ANOVA and LSD) Average overall liking scores indicated above each sample bar Plain: no added resveratrol or protein, PRO 10: same sodium caseinate concentration as SC 10, PRO 40: same sodium caseinate concentration as SC 40, Resv 10: 10 mg of unencapsulated resveratrol, Resv 40: 40 mg of unencapsulated resveratrol, P+R 10: same sodium caseinate and resveratrol concentration as SC 10, P+R 40: same sodium caseinate and resveratrol concentration as SC 40, SC 10: 110 mg resveratrol microcapsule, SC 40: 439.6 mg resveratrol microcapsules

6.67 6.546.24

6.54 6.52 6.64

6.07

6.93

6.07

1

2

3

4

5

6

7

8

9


7.056.57

7.306.90

6.236.45

6.967.48

7.00

1

2

3

4

5

6

7

8

9


A)

B)

ab ab ab ab ab ab b b a

abc bc ab abc c c abc a abc

129

Figure 6.5: Correspondence analysis from check-all-that-apply questions on attributes that are liked and disliked for A) Bars LIKE, B) Bars LIKE (zoomed in on central cluster) C) Bars LIKE for only significant attributes according to Cochran’s Q equation, D) Bars DISLIKE, E) Bars DISLIKE for only significant attributes according to Cochran’s Q equation F) Gummies LIKE, G) Gummies LIKE for only significant attributes according to Cochran’s Q equation, H) Gummies DISLIKE, I) Gummies DISLIKE for only significant attributes according to Cochran’s Q equation

A)

130

BitterMouth coating

Peanut butter flavor

Sweet

-1.5

-1

-0.5

0

0.5

1

1.5

-1.5 -1 -0.5 0 0.5 1 1.5

F2

(2

7.6

4 %

)

F1 (67.10 %)

B)

C)

Figure 6.5 (cont.)

131

Astringent

Bitter

Fatty

Sour

Sweet

-1

-0.5

0

0.5

1

-1 -0.5 0 0.5 1

F2

(1

5.8

8 %

)

F1 (75.04 %)

E)

D)

Figure 6.5 (cont.)

132

Figure 6.5 (cont.)

Chalky

Chewy

Mouth coating

Smooth

-1

-0.5

0

0.5

1

-1 -0.5 0 0.5 1

F2

(2

0.0

8 %

)

F1 (63.04 %)

F)

G)

133

ChalkyFoamy

Hard

Mouth coating

Sweet

-1

-0.5

0

0.5

1

-1 -0.5 0 0.5 1

F2

(3

0.8

0 %

)

F1 (59.08 %)

H)

I)

Figure 6.5 (cont.)

134

Figure 6.6: Prior knowledge of resveratrol health benefits of panelists who participated in

consumer testing of products with added resveratrol


Figure 6.7: Effect of health claim related to resveratrol on purchase intent of product for panelists who participated in consumer testing of products with added resveratrol


0

5

10

15

20

25

30

35

40

45

Nu

mb

er

of

Pan

elists

wh

o s

ele

cte

d

healt

h b

en

efi

t

0

5

10

15

20

25

30

35

Very likely Likely Somewhatlikely

No Effect Somewhatlikely

Unlikely Veryunlikely

Nu

mb

er

of

pan

elists

wh

o s

ele

cte

d

pu

rch

ase i

nte

nt

op

tio

n

135

Table 6.1: Formulation of bar and gummy samples used in consumer testing of products with added resveratrol

Bar (per serving)

8 g oatmeal (Meijer®, Grand Rapids, MI)

6 g milk chocolate chips (Nestlé®, Vevey, Canton of Vaud, Switzerland)

11 g peanut butter (Skippy®, Austin, MN)

6 g almond milk (Meijer®, Grand Rapids, MI)

Gummy (per serving)

2.95 g gelatin (Knox®, Kraft Foods, Chicago, IL)

2.25 g kool-aid (Kool-Aid®, Kraft Foods, Chicago, IL)

3.9 g jello (Jell-O®, Kraft Foods, Chicago, IL)

15.9 g distilled water (Meijer®, Grand Rapids, MI)

Serving size of bars are 31 g and serving size of gummies are 25 g.

136

Table 6.2: Sample codes and amount of unencapsulated resveratrol, sodium caseinate, and encapsulated resveratrol added to each serving in consumer testing of food products with added resveratrol

Sample Code Unencapsulated

resveratrol Sodium Caseinate

Encapsulated Resveratrol

Plain -- -- --

PRO 10 -- 100 mg --

PRO 40 -- 399.6 mg --

Resv 10 10 mg -- --

Resv 40 40 mg -- --

P+R 10 10 mg 100 mg --

P+R 40 40 mg 399.6 mg --

SC 10 -- -- 110 mg

SC 40 -- -- 439.6 mg

Plain: no added resveratrol or protein, PRO 10: same sodium caseinate concentration as SC 10, PRO 40: same sodium caseinate concentration as SC 40, Resv 10: 10 mg of unencapsulated resveratrol, Resv 40: 40 mg of unencapsulated resveratrol, P+R 10: same sodium caseinate and resveratrol concentration as SC 10, P+R 40: same sodium caseinate and resveratrol concentration as SC 40, SC 10: 110 mg resveratrol microcapsule, SC 40: 439.6 mg resveratrol microcapsules.

137

Table 6.3: Number of panelists in each cluster (N), average overall liking scores of SC 10 and SC 40 according to cluster analysis of overall liking scores of products with resveratrol microcapsules

Bars Gummies

Cluster 1 N = 23 SC 10 average = 7.0 SC 40 average = 7.5

N = 28 SC 10 average = 6.9 SC 40 average = 6.1





Panelists were clustered according to their overall liking scores of SC 10 and SC 40, separately for bars and gummies. SC 10: 110 mg resveratrol microcapsule, SC 40: 439.6 mg resveratrol microcapsules. There were a total of 100 panelists who participated in the consumer testing.

138

Table 6.4: Overall liking of samples for each cluster of panelists for A) Bars and B)

Gummies

A)

Sample Code Cluster 1 Cluster 2 Cluster 3

Plain 7.05abc 5.64a 4.85a

PRO 10 6.57bc 5.98a 4.69ab

PRO 40 7.30ab 5.88a 5.54a

Resv 10 6.90abc 5.93a 4.82ab

Resv 40 6.23c 5.43ab 2.92cd

P+R 10 6.45c 5.71a 5.00a

P+R 40 6.96abc 5.05bc 3.58bc

SC 10 7.48a 5.61a 3.15cd

SC 40 7.00abc 4.77c 2.31d

B)

Sample Code Cluster 1 Cluster 2 Cluster 3

Plain 6.67ab 5.11ab 5.37ab

PRO 10 6.54ab 5.19ab 5.41a

PRO 40 6.24ab 4.96ab 4.68bc

Resv 10 6.54ab 5.60a 5.05abc

Resv 40 6.52ab 5.68a 5.03abc

P+R 10 6.64ab 4.93ab 5.00abc

P+R 40 6.07b 5.44ab 4.78abc

SC 10 6.93a 4.71b 3.64d

SC 40 6.07b 2.07c 4.64c

Panelists were clustered according to their ratings of SC 10 and SC 40 using agglomerative cluster analysis. Same letters represent not significantly different values (p<0.05, ANOVA and LSD). Plain: no added resveratrol or protein, PRO 10: same sodium caseinate concentration as SC 10, PRO 40: same sodium caseinate concentration as SC 40, Resv 10: 10 mg of unencapsulated resveratrol, Resv 40: 40 mg of unencapsulated resveratrol, P+R 10: same sodium caseinate and resveratrol concentration as SC 10, P+R 40: same sodium caseinate and resveratrol concentration as SC 40, SC 10: 110 mg resveratrol microcapsule, SC 40: 439.6 mg resveratrol microcapsules.

139

Table 6.5: P values for each attribute from Cochran’s Q equation which determines if a significant difference exists across the number of times an attribute was chosen in the check-all-that-apply questions on ballot of LIKE and DISLIKE across products as evaluated in consumer testing of A) bars and B) gummies with added resveratrol

GUMMY LIKE DISLIKE

Astringent 0.310 0.703

Bitter 0.473 0.125

Chalky *0.018 *<0.001

Chewy *0.047 0.269

Foamy 0.126 *0.003

Fruity 0.104 0.699

Hard 0.074 *0.019

Mouth Coating *0.004 *0.004

Rubbery 0.394 0.099

Salty 0.721 0.084

Smooth *0.001 0.389

Soft 0.604 0.555

Sour 0.381 0.286

Sticky 0.528 0.064

Sweet 0.190 *0.002

*Significant values (α=0.05) are shown in bold

BAR LIKE DISLIKE

Astringent 0.433 *0.003

Bitter *0.042 *<0.001

Chalky 0.055 0.154

Chewy 0.395 0.336

Chocolate Flavor 0.285 0.232

Cohesive 0.362 0.510

Fatty 0.903 *0.006

Hard 0.226 0.633

Moist 0.120 0.066

Mouth Coating *0.027 0.412

Oaty 0.255 0.065

Peanut butter Flavor *0.005 0.742

Salty 0.553 0.633

Soft 0.389 0.452

Sour 0.473 *0.029

Sticky 0.201 0.076

Sweet *0.004 *0.034

Tooth Packing 0.881 0.825

A)

B)

140

Table 6.6: Nonparametric LSD results for check-all-that-apply questions showing differences across the number of times attributes were chosen across samples for A) Bars and B) Gummies, as evaluated by consumer testing on food products with added resveratrol A) LIKE DISLIKE

Sample Bitter Mouth Coating

Peanut Butter Flavor

Sweet Astringent Bitter Fatty Sour Sweet

Plain 1B 19A 64ABC 54AB 4BCD 13CD 15ABC 0B 8A

PRO 10 2AB 6B 66ABC 47AB 0D 8D 14ABC 2AB 8A

PRO 40 0B 13AB 59C 58A 5BCD 8D 6C 1B 1B

Resv 10 0B 12AB 75A 57A 3BCD 10D 13ABC 2AB 4AB

Resv 40 5A 10B 62ABC 43B 8AB 31B 21A 2AB 5AB

P+R 10 1B 10B 67ABC 53AB 2CD 11D 14ABC 3AB 8A

P+R 40 1B 9B 56C 42B 6ABC 31B 11BC 6A 5AB

SC 10 0B 11AB 73AB 51AB 4BCD 22BC 19AB 1B 4AB

SC 40 2AB 8B 60BC 41B 11A 49A 11BC 6A 2AB

B) LIKE DISLIKE

Sample Chalky Chewy Mouth

Coating Smooth Chalky Foamy Hard Mouth

Coating Sweet

Plain 1B 41AB 6ABC 31ABC 6B 11AB 11BC 3C 13A

PRO 10 1B 39AB 11AB 30ABC 4B 8B 14ABC 7BC 9AB

PRO 40 4A 30B 4BC 24BCD 26A 12AB 15ABC 12AB 7ABC

Resv 10 1B 37AB 8ABC 33AB 11B 8B 16ABC 10ABC 3BC

Resv 40 1B 45A 13A 37A 7B 8B 7C 7BC 6BC

P+R 10 1B 37AB 6ABC 30ABC 9B 8B 16ABC 18A 3BC

P+R 40 1B 38AB 9ABC 19CD 26A 18A 11BC 9BC 5BC

SC 10 1B 44A 4BC 26ABCD 23A 9B 19AB 13AB 6BC

SC 40 3A 30B 2C 17D 32A 19A 23A 13AB 2C

Same letters represent not significantly different values (p<0.05, ANOVA and LSD)

141

6.7 References





5. Opipari, A.W., et al., Resveratrol-induced autophagocytosis in ovarian cancer cells. Cancer research, 2004. 64(2): p. 696-703.






11. Schneider, Y., et al., Resveratrol inhibits intestinal tumorigenesis and modulates host-defense-related gene expression in an animal model of human familial adenomatous polyposis. Nutrition and cancer, 2001. 39(1): p. 102-107.

12. Tseng, S.H., et al., Resveratrol suppresses the angiogenesis and tumor growth of gliomas in rats. Clinical Cancer Research, 2004. 10(6): p. 2190-2202.

13. Garvin, S., K. Öllinger, and C. Dabrosin, Resveratrol induces apoptosis and inhibits angiogenesis in human breast cancer xenografts in vivo. Cancer letters, 2006. 231(1): p. 113-122.




17. Amerine, M.A., R.M. Pangborn, and E.B. Roessler, Principles of sensory evaluation of food. Principles of sensory evaluation of food., 1965.

18. Lawless, H. and H. Heymann, Sensory evaluation of food principles and practices. 1999: Springer Science + Business Media, LLC. 842.

19. Jaeger, S.R., et al., Investigation of bias of hedonic scores when co-eliciting product attribute information using cata questions. Food quality and preference, 2013. 30(2): p. 242-249.

20. Prescott, J., S.M. Lee, and K.-O. Kim, Analytic approaches to evaluation modify hedonic responses. Food quality and preference, 2011. 22(4): p. 391-393.

142

21. Jaeger, S.R. and G. Ares, Lack of evidence that concurrent sensory product characterisation using cata questions bias hedonic scores. Food quality and preference, 2014. 35: p. 1-5.


23. Ghanim, H., et al., A resveratrol and polyphenol preparation suppresses oxidative and inflammatory stress response to a high-fat, high-carbohydrate meal. The Journal of Clinical Endocrinology & Metabolism, 2011. 96(5): p. 1409-1414.

24. Green, B.G., et al., Taste mixture interactions: Suppression, additivity, and the predominance of sweetness. Physiology & behavior, 2010. 101(5): p. 731-737.

25. Tamamoto, L.C., S.J. Schmidt, and S.Y. Lee, Sensory profile of a model energy drink with varying levels of functional ingredients—caffeine, ginseng, and taurine. Journal of Food Science, 2010. 75(6): p. S271-S278.

26. Hewson, L., M. Ng, and J. Hort, Caffeine perception, effects of matrix complexity, and individual sensitivity. Journal of Caffeine Research, 2012. 2(3): p. 117-122.

27. Marshall, K., et al., The capacity of humans to identify components in complex odor–taste mixtures. Chemical senses, 2006. 31(6): p. 539-545.

28. Laing, D.G., et al., The limited capacity of humans to identify the components of taste mixtures and taste-odour mixtures. PERCEPTION-LONDON-, 2002. 31(5): p. 617-636.

143

CHAPTER 7: SUMMARY

Bioactive compounds, such as resveratrol, can help to prevent and alleviate certain

disease states. The incorporation of these compounds in food products can provide convenient

means to deliver the health benefits to consumers. Microencapsulation is an innovative

processing method that can help to stabilize the compounds during processing, storage, and

digestion, and minimize negative sensory properties of the compounds.

Whey protein and sodium caseinate were compared as encapsulation materials

evaluated by UVA light testing and a 3-stage in-vitro digestion model. After exposure to UVA

light, the trans:cis resveratrol ratios of samples were compared and a higher trans:cis

resveratrol ratio indicated higher UVA light stability. Sodium caseinate as a wall material for

encapsulation enhanced UVA light stability of resveratrol (0.63 trans:cis resveratrol ratio) in

comparison to whey protein concentrate (0.43 trans:cis resveratrol ratio) and unencapsulated

resveratrol (0.49 trans:cis resveratrol ratio). In addition, stability through the human digestive

system evaluated by digestive stability and bioaccessibility of sodium-caseinate-based

microcapsules, whey-protein-concentrate-based microcapsules and unencapsulated resveratrol

were 84% and 60%, 70% and 53%, and 47% and 23%, respectively. The addition of anhydrous

milk fat in the formulation did not have a significant effect on the stability of resveratrol within the

microcapsule. The addition of plasticizers to the microcapsule formulations caused a decrease

in UV stability of resveratrol (0.39 trans:cis resveratrol ratio) compared to the original

microcapsule formulation (0.64 trans:cis resveratrol ratio). The encapsulation of resveratrol also

helped to decrease the detection of resveratrol. The taste threshold of encapsulated resveratrol

(313-334 mg resveratrol/L) was significantly higher than that of the unencapsulated resveratrol

(90 mg resveratrol/L). In terms of consumer acceptance of products with added resveratrol, a

segment of consumers was identified whose overall liking scores of both bars and gummies with

encapsulated resveratrol were not significantly different from the plain samples (without any

144

resveratrol). Therefore, overall liking of the products was maintained when encapsulated

resveratrol was added to the products.

Stabilization of resveratrol was achieved through microencapsulation within a protein

matrix using spray drying. In the food industry, spray drying is a common technique and the

equipment is readily available to make the scale up and production of resveratrol microcapsules

feasible. In addition, the relatively low cost of protein helps to minimize the cost of the

encapsulation, thereby minimizing the additional cost of providing a stabilized form of resveratrol

to the consumer.

The developed encapsulation system is valuable as it can serve as a model into which

other components can be incorporated, such as bioactive polyphenols such as quercetin,

micronutrients such as iron, probiotics and flavor compounds for protection and controlled

release. Future research can also compare the ability of other types of proteins, such as soy

protein and pea protein, to stabilize resveratrol utilizing encapsulation. These proteins are

more suitable for individuals who are vegan or have dietary sensitivities to dairy foods. Future

research could also evaluate taste detection thresholds of resveratrol within food products as

these levels may be different than those in aqueous solutions. It would also be interesting to

add the resveratrol microcapsules to other complex food products, chocolate and protein

shakes, and evaluate consumer acceptance of these products. Consumer testing could also be

completed on food products with added resveratrol, with and without the information regarding

health benefits of resveratrol. In this way, the effect of the information about resveratrol on

consumer acceptance of the product could be determined. In addition, further research can

utilize descriptive analysis to evaluate product attribute intensities in order to confirm CATA

question results regarding attributes related to LIKE and DISLIKE attributes.

145

CHAPTER 8: APPENDICES

Appendix A: Apparent solubility of 180 mg Resveratrol/L in 1.2% Ethanol Solution


Amount Detected in Sample 181

Standard Deviation 9.3

146

Appendix B: Post-Questionnaire for Threshold Testing of Resveratrol

DEMOGRAPHICS QUESTIONNAIRE We want to ask you a few questions about yourself. This information will help us compare opinions of people with different backgrounds. All information is confidential and will not be identified with your name. You may choose not to answer questions, if you wish, as your participation is voluntary.

1. How old are you?

18-25 years old

26-35 years old

36-45 years old

46-55 years old

56-65 years old

Over 65 years old

2. What is your gender?

Male

Female

3. How do you describe yourself? (check all that apply)

American Indian or Alaska Native

Asian

Black or African American

Caucasian

Hispanic or Latino

Native Hawaiian or Other Pacific Islander

Other:

147

Appendix B (cont.) 4. When purchasing a food product which of the following criteria are most important to you?

(Rank from 1 = most important to 5 = least important, no ties)

____ Convenience ____ Health benefits

____ Nutrient content ____ Price ____ Taste

5. What product would be best aligned with the health claim “May decrease cancer risk, increase heart health and neurological function”? (Rank from 1 = most important to 4 = least important, no ties)

____ Snack Bar ____ Yogurt

____ Cookie ____ Drink

6. Which of the following health benefits do you associate with resveratrol? (Check all that apply)

Anti-aging

Improved Neurological Function




Prevent/treat cardiovascular diseases

Skin health

I am not aware of any of the health benefits associated with resveratrol


Other:

148

Appendix C: Ballot for Consumer Testing on Food Products with Resveratrol

OVERALL ACCEPTANCE OF NUTRACEUTICAL BARS

Instructions: 1. Before each sample please rinse in the following manner:

a. Rinse your mouth with warm water. b. Rinse your mouth with carbonated water. c. Rinse your mouth with room temperature water.

2. Check to ensure that the 3-digit code on the sample cup matches the one written in above the question.

3. Repeat the rinse procedure between each sample and evaluate the samples in the order they are presented on this page.

Sample Number: 506

How much do you like this sample overall?

1 2 3 4 5 6 7 8 9

Dislike

extremely

Neither

like nor

dislike

Like

extremely

What attributes of the product do you LIKE? (check all that apply) Peanut butter flavor

Bitter

Sour

Astringent

Soft

Salty

Hard

Tooth packing

Cohesive

Sticky

Moist

Mouth coating

Chewy

Fatty

Oaty

Chocolate flavor

Chalky

Sweet

149

Appendix C (cont.)

What attributes of the product do you DISLIKE? (check all that apply)

Peanut butter flavor

Bitter

Sour

Astringent

Soft

Salty

Hard

Tooth packing

Cohesive

Sticky

Moist

Mouth coating

Chewy

Fatty

Oaty

Chocolate flavor

Chalky

Sweet

Other Comments:

PLEASE RINSE NOW: First with warm water, then with carbonated water, and then with

room temperature water.

150

Appendix D: Post-Questionnaire for Consumer Testing on Food Products with Resveratrol

DEMOGRAPHICS QUESTIONNAIRE

We want to ask you a few questions about yourself. This information will help us compare opinions

of people with different backgrounds. All information is confidential and will not be identified with

your name. You may choose not to answer questions, if you wish, as your participation is

voluntary.

1. How old are you?

18-25 years old

26-35 years old

36-45 years old

46-55 years old

56-65 years old

Over 65 years old

2. What is your gender?

Male

Female

3. How do you describe yourself? (check all that apply)

American Indian or Alaska Native

Asian

Black or African American

Caucasian

Hispanic or Latino

Native Hawaiian or Other Pacific Islander

Other:

151

Appendix D (cont.)

4. Which of the following health benefits do you associate with resveratrol? (Check all that

apply)

Anti-aging

Improved Neurological Function




Prevent/treat cardiovascular diseases

Skin health

I am not aware of any of the health benefits associated with resveratrol


Other:

5. If a product was labeled with “May decrease cancer risk, increase heart health and

neurological function”, would this increase the chance that you purchase this product over

another product without a health claim?

Very Likely

Likely

Somewhat Likely

No Effect

Somewhat Unlikely

Unlikely

Very unlikely

You have now completed the test. Thank you for your participation.

Please return your ballot.

Documents

STABILIZATION OF RESVERATROL THROUGH …